Systems, methods, and workflows for optogenetics analysis

ABSTRACT

The invention provides methods for characterizing cellular physiology by incorporating into an electrically excitable cell an optical reporter of, and an optical actuator of, electrical activity. A signal is obtained from the optical reporter in response to a stimulation of the cell. Either or both of the optical reporter and actuator may be based on genetically-encoded rhodopsins incorporated into the cell. The invention provides all optical methods that may be used instead of, or as a complement to, traditional patch clamp technologies and that can provide rapid, accurate, and flexible assays of cellular physiology.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a continuation of and claims priority under 35U.S.C. §120 to U.S. patent application, U.S. Ser. No. 14/303,178, filedJun. 12, 2014, which is a continuation-in-part of U.S. patentapplication, U.S. Ser. No. 13/818,432, filed May 13, 2013, which is anational stage filing under 35 U.S.C. §371 of international PCTapplication, PCT/US2011/048793, filed Aug. 23, 2011, which claimspriority under 35 U.S.C. §119(e) to U.S. provisional patentapplications, U.S. Ser. No. 61/376,049, filed Aug. 23, 2010 and U.S.Ser. No. 61/412,972, filed Nov. 12, 2010, the contents of each of whichare incorporated herein by reference in their entirety.

FIELD OF INVENTION

The invention relates to methods of characterizing the electrophysiologyof cells.

BACKGROUND

In the late 1700s, Luigi Galvani observed a frog carcass move inresponse to electric stimulus. This led to the insight that cells areaffected by and can transmit electrical signals. Two hundred yearslater, the patch clamp provided a way to study the electrical physiologyof cells. See, e.g., Liem et al., 1995, The patch clamp technique,Neurosurgery 36(2):382-92.

The patch clamp technique can potentially illustrate processes such assignal transduction and synaptic transmission as well as consequences ofconditions such as demyelination, brain and spinal cord injury, andcardiac arrhythmias—to give but a few examples. Unfortunately, thetechnique is remarkably difficult and time-consuming to implement. Toset up a patch clamp, the tip of a glass pipette must be pushed againsta patch of cell membrane and suction applied to create a clamp betweenthe pipette and the patch. The patch ruptures, cytosol enters thepipette, and measurements are made in the pipette to determine themembrane electrical properties. Variations of this basic theme areknown, but they all require the fundamentally difficult and limitingsteps of physically clamping a pipette to a patch of cell membrane.

SUMMARY

The invention provides methods for characterizing cellular physiology byincorporating into one or more electrically excitable cells an opticalreporter of, and an optical actuator of, electrical activity. Theactuator and reporter can be in the same cell or in different cells thatare functionally coupled (e.g. by synapses or electrical gap junctions).A signal is obtained from the optical reporter in response to astimulation of the actuator. Either or both of the optical reporter andactuator may be based on genetically-encoded microbial rhodopsins whosegenes have been incorporated into the cell. The invention providesall-optical methods that may be used instead of, or as a complement to,traditional patch clamp technologies and that can provide rapid,accurate, and flexible assays of cellular physiology and synapticfunction.

The invention further includes methods for converting a somatic cellsuch as a fibroblast into a specific type of electrically excitable cellusing stem cell or lineage conversion technologies. A sample may betaken from a patient or other subject and converted into a neuron, acardiomyocyte, a glial cell, any other suitable cell type, and even intospecific cellular sub-types such as a motor neuron. The inventionadditionally provides for genome editing, so that variants of a genomeof a sample or subject may be studied. By these means, a condition knownto affect a certain tissue type may be studied, or the cellularphenotype of a certain mutation as well as the wild-type allele may bestudied. The “optical patch clamp” methods of the invention provide fora rapid and productive way to study cellular physiology. Using methodsof the invention, conditions may be diagnosed, drugs tested, andcellular function may be illustrated. This gives practitioners valuabletools for studying processes such as signal transduction and synaptictransmission as well as consequences of conditions such asdemyelination, brain and spinal cord injury, and cardiac arrhythmias.

In certain aspects, the invention provides a method for characterizing acell by incorporating into an electrically excitable cell an opticalreporter of electrical activity. An optical actuator of electricalactivity may also be incorporated, either into the cell or into anothercell (e.g., one that is in synaptic connection with the cell). A signalis obtained from the optical reporter in response to a stimulation ofthe cell. The cell is characterized by evaluating the signal.

The observed signal may comprise a probability of a voltage spike inresponse to the stimulation of the cell, for example, a changedprobability of a voltage spike in response to the stimulation of thecell relative to a control. The observed signal may also comprise achange in the waveform of a voltage spike; a change in the propagationof a voltage spike; or a sub-threshold excitatory or inhibitory changein membrane voltage that does not comprise a voltage spike.Characterizing the cell may include evaluating a response of the cell toexposure to a compound; measuring a concentration of an ion; determiningprogress of a treatment; or diagnosing a disease. A disease such asCockayne syndrome, Down Syndrome, Dravet syndrome, familialdysautonomia, Fragile X Syndrome, Friedreich's ataxia, Gaucher disease,hereditary spastic paraplegias, Machado-Joseph disease, Phelan-McDermidsyndrome (PMDS), polyglutamine (polyQ)-encoding CAG repeats, spinalmuscular atrophy, Timothy syndrome, Alzheimer's disease, frontotemporallobar degeneration, Huntington's disease, multiple sclerosis,Parkinson's disease, spinal and bulbar muscular atrophy, or amyotrophiclateral sclerosis may be studied or diagnosed.

In certain embodiments, the actuator, reporter, or both are incorporatedby transforming the electrically active cell with a vector that includesa nucleic acid encoding the optical actuator of electrical activity, theoptical reporter of electrical activity, or both. Thus one or any numberof individual cells may include both an optical actuator of, and anoptical reporter of, electrical activity. Alternatively, an opticalactuator may be incorporated into one or any number of individual cellsand an optical reporter may be incorporated into one or any number ofother cells (e.g., cell or cells in synaptic connection with thereporter cell or cells).

The actuator and reporter may be incorporated into different cells forapplications such as studying synaptic transmission and networkproperties in cultured neurons. Transmission is also important forcharacterizing the strength of the gap junction connections in cardiaccultures.

The optical actuator may be a genetically-encoded rhodopsin or modifiedrhodopsin such as a microbial channelrhodopsin. For example, sdChR, achannelrhodopsin from Scherffelia dubia, may be used or an improvedversion of sdChR—dubbed CheRiff—may be used as an optical actuator.“CheRiff” refers to a version of sdChR that uses mouse codonoptimization, a trafficking sequence, and the mutation E154A asdescribed herein.

The optical reporter may be a genetically-encoded rhodopsin or modifiedrhodopsin such as a microbial rhodopsin that exhibits fluorescence. Forexample modified versions of the microbial rhodopsin proteinArchaerhodopsin 3 (Arch) from Halorubum sodomense may be used.

In some embodiments, the method includes obtaining a somatic cell andconverting the somatic cell into the electrically excitable cell. Asomatic cell may be converted into the electrically active cell bydirect conversion or via an induced pluripotent stem cell (iPS)intermediary or an embryonic stem cell.

The optical actuator may initiate an action potential in response to thestimulation (e.g., illuminating the cell). The cell can be illuminatedusing an instrument of the invention that provides spatially resolvedlight from a digital micromirror array or a spatial light modulator. Insome embodiments, the excitation and emission wavelengths of the opticalreporter comprise light that does not stimulate the cell. Since thereporter wavelengths and actuator wavelength can be spectrallyorthogonal, the invention provides methods for illuminating the cell andobtaining the signal simultaneously. Methods may include obtaining acontrol cell and observing a control signal generated by a controloptical reporter in the control cell. Obtaining the control cell mayinclude editing a genome from the cell such that the control cell andthe cell are isogenic but for a mutation.

The invention includes methods for processing the observed signal orsignals and resolving single-cells signals from among numerous signalsfrom spatially overlapping cells. Obtaining the signal may includeobserving a cluster of different cells with a microscope and using acomputer to isolate the signal generated by the optical reporter from aplurality of signals from the individual cells. The computer isolatesthe signal by performing an independent component analysis andidentifying a spike train associated with the cell. A microscope of theinvention may be used to obtain an image of a plurality of clusters ofcells. Unlike other systems that require one image per cluster or percell, a wide field microscope system with signal deconvolution can imageand distinguish a plurality of cells or clusters per image.

Aspects of the invention provide a method for characterizing aninteraction between cells. The method includes incorporating into afirst electrically excitable cell an optical actuator of electricalactivity, incorporating into a second electrically excitable cell anoptical reporter of electrical activity, and culturing the firstelectrically excitable cell and the second electrically excitable cellin proximity to one another. A signal is obtained from the opticalreporter in response to a stimulation of the first electricallyexcitable cell. The signal is evaluated, thereby characterizing aninteraction between the first electrically excitable cell and the secondelectrically excitable cell. The first electrically excitable cell andthe second electrically excitable cell may be of the same cell type ordifferent, for example, either or both may be a neuron, a cardiomyocyte,or a glial cell. The characterized interaction may be, for example,excitatory neurotransmission, inhibitory neurotransmission, or theconduction velocity of cardiac action potential. In some embodiments,incorporating the actuator or reporter into the first or second,respectively, electrically excitable cell is done by transforming firstelectrically excitable cell with a vector that includes a nucleic acidencoding the optical actuator of electrical activity. The opticalactuator may be provided by a modified rhodopsin as discussed herein.The optical reporter may be a rhodopsin that has been modified (e.g.,for voltage-sensitive fluorescence and absence of a steady-statephotocurrent). The observed signal may include a probability of avoltage spike in response to the stimulation of the cell, a changedprobability of a voltage spike in response to the stimulation of thecell relative to a control, a change in the waveform of a voltage spike,a sub-threshold increase in the membrane potential, or a decrease in themembrane potential. The characterized interaction may be applied indiagnosing a disease, evaluating a cellular response to exposure to acompound, or determining progress of a treatment.

Methods can include obtaining somatic cells and converting the somaticcells into the first electrically excitable cell and the secondelectrically excitable cell. Converting the somatic cells may be viadirect conversion, via an iPS intermediary, or the first electricallyexcitable cell and the second electrically excitable cell may be derivedfrom a human embryonic stem cell.

In certain embodiments, the optical actuator initiates an actionpotential in response to illuminating the first electrically excitablecell (e.g., done using spatially resolved light from a digitalmicromirror array). Preferably, the excitation of, and the signal from,the optical reporter comprise light that does not stimulate the firstelectrically excitable cell. Thus, the illuminating and obtaining thesignal may be done simultaneously.

In some embodiments, obtaining the signal is done by observing a clusterof different cells with a microscope and using a computer to isolate thesignal generated by the optical reporter from a plurality of signalsfrom the different cells. A microscope obtains an image of a pluralityof clusters of cells. The computer isolates the signal by performing anindependent component analysis and identifying a spike train associatedwith the second electrically excitable cell.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates a method for characterizing a cell.

FIG. 2 illustrates exemplary pathways for converting cells into specificneural subtypes.

FIG. 3 gives an overview of a method for genome editing.

FIG. 4 shows genetically encoded fluorescent voltage indicatorsclassified according to their sensitivity and speed.

FIG. 5 shows electrically recorded membrane potential of a neuronexpressing Arch WT.

FIG. 6 shows electrically recorded membrane potential of a neuronexpressing Arch D95N.

FIG. 7 shows whole-cell membrane potential determined via electricalrecording.

FIG. 8 shows optical recordings of action potentials in a single HL-1mouse cardiomyocyte expressing Arch 3 D95N-eGFP.

FIG. 9 shows optical recordings of the action potentials in a singleHL-1 cell over time.

FIG. 10 gives a functional diagram of components of an optical imagingapparatus.

FIG. 11 illustrates a pulse sequence of red and blue light used torecord action potentials.

FIG. 12 shows an image that contains five neurons whose images overlap.

FIG. 13 shows clusters of pixels whose intensity varies synchronouslyfound by an independent component analysis (ICA).

FIG. 14 illustrates contributions from individual cells to the ICA timecourse.

FIG. 15 shows an overlay of filters used to map individual cells in animage.

FIG. 16 shows a patterned optical excitation being used to induce actionpotentials.

FIG. 17 shows eigenvectors resulting from a principal component analysisof a single action potential waveform.

FIG. 18 shows a relation between cumulative variance and eigenvectornumber for the principal component analysis of FIG. 17.

FIG. 19 compares action potential waveforms before and after smoothingoperations.

FIG. 20 shows an action potential timing map.

FIG. 21 shows the accuracy of timing extracted by a sub-Nyquistaction-potential timing (SNAPT) algorithm.

FIG. 22 gives an image of eGFP fluorescence, indicating CheRiffdistribution in a neuron.

FIG. 23 presents frames from a SNAPT movie.

FIG. 24 illustrates an output from measuring action potentials inhiPSC-derived motor neurons containing a mutation associated withamyotrophic lateral sclerosis.

FIG. 25 demonstrates effects of dimethyl sulfoxide (DMSO) onhiPSC-derived cardiomyocytes action potential waveform.

FIG. 26 presents the effects of DMSO control vehicle and pacing rate onthe average action potential waveform.

FIG. 27 presents the effects of DMSO control vehicle and pacing rate onthe average rise time.

FIG. 28 shows the dose dependent response of action potential width at50% repolarization (AP50) to increasing concentrations of DMSO.

FIG. 29 shows the dose dependent response of action potential rise timeto increasing concentrations of DMSO FIG. 30 shows the dose dependentresponse of action potential width at 90% repolarization (AP90) toincreasing concentrations of DMSO.

FIG. 31 shows the dose dependence of the spontaneous beat rate as afunction of DMSO concentration.

FIG. 32 shows a model of Arch as a voltage sensor.

FIG. 33 shows absorption and fluorescence emission spectra.

FIG. 34 top shows a HEK cell expressing Arch, visualized via Archfluorescence.

FIG. 35 shows fluorescence of Arch as a function of membrane potential.

FIG. 36 shows dynamic response of Arch to steps in membrane potential.

FIG. 37 shows sensitivity of Arch 3 WT to small steps in membranevoltage.

FIG. 38 shows that Arch 3 reports action potentials without exogenousretinal.

FIG. 39 presents a system useful for performing methods of theinvention.

DETAILED DESCRIPTION

FIG. 1 illustrates a method 101 to characterize 133 a cell. Methods aregiven to obtain 107 an electrically excitable cell. An optical actuatorof, and an optical reporter of, electrical activity are incorporatedinto the cell. Preferably, the cell will express 113 (e.g., bytranslation) the reporter. An optical signal from the optical reporterin response to a stimulation of the cell is obtained. To characterizethe cell, one may observe 123 a signature of the signal and analyze orevaluate 127 the signature. By evaluating the signal, one maycharacterize 133 the cell.

Additionally, methods of the invention may be employed to study and usenetwork effects whereby one cell or genetically specified cell type isstimulated, and a different one is recorded. Both cells might have bothactuator and reporter; or one might have actuator only, and the otherreporter only. This ability to probe network effects may be particularlyimportant as many genes, such as ones that are being implicated inschizophrenia and bipolar disorder, code for synaptic proteins. Networkeffects also promise to be important in the cardiac area, where forexample a monolayer of cardiomyocytes may be illuminated with some cellsexpressing actuators of the invention while imaged via others expressingthe reporters.

1. Obtaining Cell(s)

Cells that are useful according to the invention include eukaryotic andprokaryotic cells. Eukaryotic cells include cells of non-mammalianinvertebrates, such as yeast, plants, and nematodes, as well asnon-mammalian vertebrates, such as fish and birds. The cells alsoinclude mammalian cells, including mouse, rat, and human cells. Thecells also include immortalized cell lines such as HEK, HeLa, CHO, 3T3,PC12, which may be particularly useful in applications of the methodsfor drug screens. The cells also include stem cells, embryonic stemcells, pluripotent cells, progenitor cells, and induced pluripotentcells. Differentiated cells including cells differentiated from the stemcells, pluripotent cells and progenitor cells are included as well.

Cells are obtained by any suitable means. For example, methods of theinvention can include obtaining one or more cells such as fibroblastsfrom an organism such as a person or animal. In some embodiments, adermal biopsy is performed to obtain dermal fibroblasts. The skin isanesthetized and a sterile 3 mm punch is used to apply pressure and makea drilling motion until the punch has pierced the epidermis. A biopsysample is lifted out and transferred to a sterile BME fibroblast mediumafter optional washing with PBS and evaporation of the PBS. The biopsysite on the patient is dressed (e.g., with an adhesive bandage).Suitable methods and devices for obtaining the cells are discussed inU.S. Pat. No. 8,603,809; U.S. Pat. No. 8,403,160; U.S. Pat. No.5,591,444; U.S. Pub. 2012/0264623; and U.S. Pub. 2012/0214236, thecontents of each of which are incorporated by reference. Any tissueculture technique that is suitable for the obtaining and propagatingbiopsy specimens may be used such as those discussed in Freshney, Ed.,1986, Animal Cell Culture: A Practical Approach, IRL Press, OxfordEngland; and Freshney, Ed., 1987, Culture of Animal Cells: A Manual ofBasic Techniques, Alan R. Liss & Co., New York, both incorporated byreference.

In some embodiments, the cells are cultured in vitro or ex vivo. In someembodiments, the cells are part of an organ or an organism.

In some embodiment, the cell is an “artificial cell” or a “syntheticcell” created by bioengineering (see, e.g., Gibson et al., 2010,Creation of a Bacterial Cell Controlled by a Chemically SynthesizedGenome, Science 329(5987):52-56; Cans et al., 2008, Positioning LipidMembrane Domains in Giant Vesicles by Micro-organization of AqueousCytoplasm Mimic, J Am Chem Soc 130:7400-7406.

The methods can also be applied to any other membrane-bound structure,which may not necessarily be classified as a cell. Such membrane boundstructures can be made to carry the microbial rhodopsin proteins of theinvention by, e.g., fusing the membranes with cell membrane fragmentsthat carry the microbial rhodopsin proteins of the invention.

Cells may include zebrafish cardiomyocytes; immune cells (primary murineand human cultures and iPS-derived lines for all, in addition to thespecific lines noted below), including B cells (e.g., human Raji cellline, and the DT40 chicken cell line), T cells (e.g., human Jurkat cellline), Macrophages, Dendritic cells, and Neutrophils (e.g., HL-60 line).Additionally, one can use glial cells: astrocytes and oligodendrocytes;pancreatic beta cells; hepatocytes; non-cardiac muscle cells; endocrinecells such as parafollicular and chromaffin; and yeast cells. Cellsfurther include neuronal cells, such as neurons.

The cell can also be a Gram positive or a Gram negative bacteria, aswell as pathogenic bacteria of either Gram type. The pathogenic cellsare useful for applications of the method to, e.g., screening of novelantibiotics that affect membrane potential to assist in destruction ofthe bacterial cell or that assist destruction of the bacterial cell incombination with the membrane potential affecting agent; or in thesearch for compounds that suppress efflux of antibiotics.

The membrane potential of essentially any cell, or any phospholipidbilayer enclosed structure, can be measured using the methods andcompositions described herein. Examples of the cells that can be assayedare a primary cell e.g., a primary hepatocyte, a primary neuronal cell,a primary myoblast, a primary mesenchymal stem cell, primary progenitorcell, or it may be a cell of an established cell line. It is notnecessary that the cell be capable of undergoing cell division; aterminally differentiated cell can be used in the methods describedherein. In this context, the cell can be of any cell type including, butnot limited to, epithelial, endothelial, neuronal, adipose, cardiac,skeletal muscle, fibroblast, immune cells, hepatic, splenic, lung,circulating blood cells, reproductive cells, gastrointestinal, renal,bone marrow, and pancreatic cells. The cell can be a cell line, a stemcell, or a primary cell isolated from any tissue including, but notlimited to brain, liver, lung, gut, stomach, fat, muscle, testes,uterus, ovary, skin, spleen, endocrine organ and bone, etc. Where thecell is maintained under in vitro conditions, conventional tissueculture conditions and methods can be used, and are known to those ofskill in the art. Isolation and culture methods for various cells arewell within the knowledge of one skilled in the art. The cell can be aprokaryotic cell, a eukaryotic cell, a mammalian cell or a human cell.In one embodiment, the cell is a neuron or other cell of the brain. Insome embodiment, the cell is a cardiomyocyte. In some embodiments, thecell is cardiomyocyte that has been differentiated from an inducedpluripotent cell.

2. Converting Cell(s) into Neurons, Cardiomyocytes, or Specific NeuralSub-Types

Obtained cells may be converted into any electrically excitable cellssuch as neurons, specific neuronal subtypes, astrocytes or other glia,cardiomyocytes, or immune cells. Additionally, cells may be convertedand grown into co-cultures of multiple cell types (e.g. neurons+glia,neurons+cardiomyocytes, neurons+immune cells).

FIG. 2 illustrates exemplary pathways for converting cells into specificneural subtypes. A cell may be converted to a specific neural subtype(e.g., motor neuron). Suitable methods and pathways for the conversionof cells include pathway 209, conversion from somatic cells to inducedpluripotent stem cells (iPSCs) and conversion of iPSCs to specific celltypes, or pathways 211 direct conversion of cells in specific celltypes.

2a. Conversion of Cells to iPSs and Conversion of iPSs to Specific CellTypes

Following pathways 209, somatic cells may be reprogrammed into inducedpluripotent stem cells (iPSCs) using known methods such as the use ofdefined transcription factors. The iPSCs are characterized by theirability to proliferate indefinitely in culture while preserving theirdevelopmental potential to differentiate into derivatives of all threeembryonic germ layers. In certain embodiments, fibroblasts are convertedto iPSC by methods such as those discussed in Takahashi and Yamanaka,2006, Induction of pluripotent stem cells from mouse embryonic and adultfibroblast cultures by defined factors Cell 126:663-676.; and Takahashi,et al., 2007, Induction of pluripotent stem cells from adult humanfibroblasts by defined factors, Cell 131:861-872.

Induction of pluripotent stem cells from adult fibroblasts can be doneby methods that include introducing four factors, Oct3/4, Sox2, c-Myc,and Klf4, under ES cell culture conditions. Human dermal fibroblasts(HDF) are obtained. A retroviruses containing human Oct3/4, Sox2, Klf4,and c-Myc is introduced into the HDF. Six days after transduction, thecells are harvested by trypsinization and plated onto mitomycinC-treated SNL feeder cells. See, e.g., McMahon and Bradley, 1990, Cell62:1073-1085. About one day later, the medium (DMEM containing 10% FBS)is replaced with a primate ES cell culture medium supplemented with 4ng/mL basic fibroblast growth factor (bFGF). See Takahashi, et al.,2007, Cell 131:861. Later, hES cell-like colonies are picked andmechanically disaggregated into small clumps without enzymaticdigestion. Each cell should exhibit morphology similar to that of humanES cells, characterized by large nuclei and scant cytoplasm. The cellsafter transduction of HDF are human iPS cells. DNA fingerprinting,sequencing, or other such assays may be performed to verify that the iPScell lines are genetically matched to the donor.

These iPS cells can then be differentiated into specific neuronalsubtypes. Pluripotent cells such as iPS cells are by definition capableof differentiating into cell types characteristic of different embryonicgerm layers. A property of both embryonic stem cells human iPS cells istheir ability, when plated in suspension culture, to form embryoidbodies (EBs). EBs formed from iPS cells are treated with two smallmolecules: an agonist of the sonic hedgehog (SHH) signaling pathway andretinoic acid (RA). For more detail, see the methods described in Dimoset al., 2008, Induced pluripotent stem cells generated from patientswith ALS can be differentiated into motor neurons, Science321(5893):1218-21; Amoroso et al., 2013, Accelerated high-yieldgeneration of limb-innervating motor neurons from human stem cells, JNeurosci 33(2):574-86; and Boulting et al., 2011, A functionallycharacterized test set of human induced pluripotent stem cells, NatBiotech 29(3):279-286; Davis-Dusenbery et al., 2014, How to make spinalmotor neurons, Development 141(3):491-501; Sandoe and Eggan, 2013,Opportunities and challenges of pluripotent stem cell neurodegenerativedisease models, Nat Neuroscience 16(7):780-9; and Han et al., 2011,Constructing and deconstructing stem cell models of neurologicaldisease, Neuron 70(4):626-44.

2b. Direct Conversion of Cells in Specific Cell Types

By pathway 211, human somatic cells are obtained and direct lineageconversion of the somatic cells into motor neurons may be performed.Conversion may include the use of lineage-specific transcription factorsto induce the conversion of specific cell types from unrelated somaticcells. See, e.g., Davis-Dusenbery et al., 2014, How to make spinal motorneurons, Development 141:491; Graf, 2011, Historical origins oftransdifferentiation and reprogramming, Cell Stem Cell 9:504-516. It hasbeen shown that a set of neural lineage-specific transcription factors,or BAM factors, causes the conversion of fibroblasts into inducedneuronal(iN) cells. Vierbuchen 2010 Nature 463:1035. MicroRNAs andadditional pro-neuronal factors, including NeuroD1, may cooperate withor replace the BAM factors during conversion of human fibroblasts intoneurons. See, for example, Ambasudhan et al., 2011, Direct reprogrammingof adult human fibroblasts to functional neurons under definedconditions, Cell Stem Cell 9:113-118; Pang et al., 2011, Induction ofhuman neuronal cells by defined transcription factors, Nature476:220-223; also see Yoo et al., 2011, MicroRNA mediated conversion ofhuman fibroblasts to neurons, Nature 476:228-231.

2c. Maintenance of Differentiated Cells

Differentiated cells such as motor neurons may be dissociated and platedonto glass coverslips coated with poly-d-lysine and laminin. Motorneurons may be fed with a suitable medium such as a neurobasal mediumsupplemented with N2, B27, GDNF, BDNF, and CTNF. Cells may be maintainedin a suitable medium such as an N2 medium (DMEM/F12 [1:1] supplementedwith laminin [1 μg/mL; Invitrogen], FGF-2 [10 ng/ml; R&D Systems,Minneapolis, Minn.], and N2 supplement [1%; Invitrogen]), furthersupplemented with GDNF, BDNF, and CNTF, all at 10 ng/ml. Suitable mediaare described in Son et al., 2011, Conversion of mouse and humanfibroblasts into functional spinal motor neurons, Cell Stem Cell9:205-218; Vierbuchen et al., 2010, Direct conversion of fibroblasts tofunctional neurons by defined factors, Nature 4 63:1035-1041; Kuo etal., 2003, Differentiation of monkey embryonic stem cells into neurallineages, Biology of Reproduction 68:1727-1735; and Wernig et al., 2002,Tau EGFP embryonic stem cells: an efficient tool for neuronal lineageselection and transplantation. J Neuroscience Res 69:918-24, eachincorporated by reference.

3. Control Cell Line or Signature or Reference Value

Methods of the invention may include obtaining or observing a signalfrom the cell and comparing the observed signal to an expected signal,such as a signal obtained from a reference.

The term “reference” as used herein refers to a baseline value of anykind that one skilled in the art can use in the methods. In someembodiments, the reference is a cell that has not been exposed to astimulus capable of or suspected to be capable of changing membranepotential. In one embodiment, the reference is the same cell transfectedwith the microbial rhodopsin but observed at a different time point. Inanother embodiment, the reference is the fluorescence of a homologue ofGreen Fluorescent Protein (GFP) operably fused to the microbialrhodopsin.

The reference signature may be obtained by obtaining a control cell thatis also of the specific neural subtype and is genetically andphenotypically similar to the test cells. In certain embodiments—where,for example, a patient has a known mutation or allele at a certainlocus—genetic editing is performed to correct the mutation and generatea control cell.

Genetic or genome editing techniques may proceed by any suitable methodsuch as zinc-finger domain methods, transcription activator-likeeffector nucleases (TALENs), or clustered regularly interspaced shortpalindromic repeat (CRISPR) nucleases. Genome editing may be used tocreate a control cell that is isogenic but-for a variant of interest orto obtain other variants of the original genome, such as knocking out agene, introducing a premature stop codon, interfering with a promoterregion, or changing the function of an ion channel or other cellularprotein. In certain embodiments, genome editing techniques are appliedto the iPS cells. Genomic editing may be performed by any suitablemethod known in the art such as TALENs or CRISPR technology. TALENs andCRISPR methods provide one-to-one relationship to the target sites, i.e.one unit of the tandem repeat in the TALE domain recognizes onenucleotide in the target site, and the crRNA or gRNA of CRISPR/Cassystem hybridizes to the complementary sequence in the DNA target.Methods can include using a pair of TALENs or a Cas9 protein with onegRNA to generate double-strand breaks in the target. The breaks are thenrepaired via non-homologous end joining or homologous recombination(HR).

TALENs uses a nonspecific DNA-cleaving nuclease fused to a DNA-bindingdomain that can be to target essentially any sequence. For TALENtechnology, target sites are identified and expression vectors are made.See Liu et al, 2012, Efficient and specific modifications of theDrosophila genome by means of an easy TALEN strategy, J. Genet. Genomics39:209-215. The linearized expression vectors (e.g., by Not1) and usedas template for mRNA synthesis. A commercially available kit may be usesuch as the mMESSAGE mMACHINE SP6 transcription kit from LifeTechnologies (Carlsbad, Calif.). See Joung & Sander, 2013, TALENs: awidely applicable technology for targeted genome editing, Nat Rev MolCell Bio 14:49-55.

CRISPR methodologies employ a nuclease, CRISPR-associated (Cas9), thatcomplexes with small RNAs as guides (gRNAs) to cleave DNA in asequence-specific manner upstream of the protospacer adjacent motif(PAM) in any genomic location. CRISPR may use separate guide RNAs knownas the crRNA and tracrRNA. These two separate RNAs have been combinedinto a single RNA to enable site-specific mammalian genome cuttingthrough the design of a short guide RNA. Cas9 and guide RNA (gRNA) maybe synthesized by known methods. Cas9/guide-RNA (gRNA) uses anon-specific DNA cleavage protein Cas9, and an RNA oligo to hybridize totarget and recruit the Cas9/gRNA complex. See Chang et al., 2013, Genomeediting with RNA-guided Cas9 nuclease in zebrafish embryos, Cell Res23:465-472; Hwang et al., 2013, Efficient genome editing in zebrafishusing a CRISPR-Cas system, Nat. Biotechnol 31:227-229; Xiao et al.,2013, Chromosomal deletions and inversions mediated by TALENS andCRISPR/Cas in zebrafish, Nucl Acids Res 1-11.

In certain embodiments, genome editing is performed using zinc fingernuclease-mediated process as described, for example, in U.S. Pub.2011/0023144 to Weinstein.

FIG. 3 gives an overview of a method 301 for zing-finger nucleasemediated editing. Briefly, the method includes introducing into the iPScell at least one RNA molecule encoding a targeted zinc finger nuclease305 and, optionally, at least one accessory polynucleotide. The cellincludes target sequence 311. The cell is incubated to allow expressionof the zinc finger nuclease 305, wherein a double-stranded break 317 isintroduced into the targeted chromosomal sequence 311 by the zinc fingernuclease 305. In some embodiments, a donor polynucleotide or exchangepolynucleotide 321 is introduced. Target DNA 311 along with exchangepolynucleotide 321 may be repaired by an error-prone non-homologous endjoining DNA repair process or a homology-directed DNA repair process.This may be used to produce a control line with a control genome 315that is isogenic to original genome 311 but for a changed site. Thegenomic editing may be used to establish a control line (e.g., where thepatient is known to have a certain mutation, the zinc finger process mayrevert the genomic DNA to wild type) or to introduce a mutation (e.g.,non-sense, missense, or frameshift) or to affect transcription orexpression. See also Beerli & Barbas, 2002, Engineering polydactylzing-finger transcription factors, Nat. Biotechnol, 20:135-141; Pabo etal., 2001, Design and selection of novel Cys2His2 zinc finger proteins,Ann. Rev. Biochem 70:313-340; Isalan et al., 2001, A rapid generallyapplicable method to engineer zinc fingers illustrated by targeting theHIV-1 promoter, Nat. Biotechnol 19:656-660; Santiago et al., 2008,Targeted gene knockout in mammalian cells by using engineeredzinc-finger nucleases, PNAS 105:5809-5814; Santiago et al, 2008,Targeted gene knockout in mammalian cells by using engineered zincfinger nucleases, PNAS 105:5809-5814; Moehle et al., 2007, Targeted geneaddition into a specified location in the human genome using designedzinc finger nucleases PNAS 104:3055-3060; Urnov et al., 2005, Highlyefficient endogenous human gene correction using designed zinc-fingernucleases, Nature 435(7042):646-51; and Lombardo et al., 2007, Geneediting in human stem cells using zinc finger nucleases andintegrase-defective lentiviral vector delivery, Nat Biotechnol25(11):1298-306; U.S. Pat. Nos. 6,607,882; 6,534,261 and 6,453,242; U.S.Pat. No. 5,789,538; U.S. Pat. No. 5,925,523; U.S. Pat. No. 6,007,988;U.S. Pat. No. 6,013,453; U.S. Pat. No. 6,410,248; U.S. Pat. No.6,140,466; U.S. Pat. No. 6,200,759; and U.S. Pat. No. 6,242,568, each ofwhich is incorporated by reference.

Using genome editing for modifying a chromosomal sequence, a controlcell or cell line can be generated, or any other genetic variant of thefirst cell may be created. In certain embodiments, control cells areobtained from healthy individuals, i.e., without using genome editing oncells taken from the subject. The control line can be used to generate acontrol signature, or reference, for comparison to test data. In someembodiments, a control signature is stored on-file after having beenpreviously generated and stored and the stored control signature is used(e.g., a digital file such as a graph or series of measurements storedin a non-transitory memory in a computer system). For example, a controlsignature could be generated by assaying a large population of subjectsof known phenotype or genotype and storing an aggregate result as acontrol signature for later downstream comparisons.

4. Optogenetic Systems

In a preferred embodiment, methods of the invention includecharacterizing a cell by incorporating into a cell an optical actuatorof electrical activity and an optical reporter of electricalactivity—i.e., both into one cell or each of a plurality of cells. Insome embodiments, a cell will receive one of the actuator and reporter.In certain embodiments, a cell will receive both via transfection with asingle vector that includes genes coding for each of the reporter andactuator. As used herein the term “optical reporter” refers to astructure or system employed to yield an optical signal indicative ofcellular electrical or neural activity such as a voltage drop across amembrane, a synaptic transmission, an action potential, a release oruptake or non-uptake of a neurotransmitter, etc. As used herein, theterm “membrane potential” refers to a calculated difference in voltagebetween the interior and exterior of a cell. In one embodiment membranepotential, ΔV, is determined by the equation ΔV=V(interior)−V(exterior).By convention, V(exterior) is regarded as 0 V, so then ΔV=V(interior).

4a. Optogenetic Reporters

The cell and the optional control line may be caused to express anoptical reporter of neural or electrical activity. Examples of neuralactivity include action potentials in a neuron or fusion of vesiclesreleasing neurotransmitters. Exemplary electrical activity includesaction potentials in a neuron, cardiomyocyte, astrocyte or otherelectrically active cell. Further examples of neural or electricalactivity include ion pumping or release or changing ionic gradientsacross membranes. Causing a cell to express an optical reporter ofneural activity can be done with a fluorescent reporter of vesiclefusion. Expressing an optical reporter of neural or electrical activitycan include transformation with an optogenetic reporter. For example,the cell may be transformed with a vector comprising an optogeneticreporter and the cell may also be caused to express an optogeneticactuator by transformation. In certain embodiments, the differentiatedneurons are cultured (e.g., for about 4 days) and then infected withlentivirus bearing a genetically encoded optical reporter of neural orelectrical activity and optionally an optical voltage actuator.

Any suitable optical reporter of neural or electrical activity may beused. Exemplary reporters include fluorescent reporters of transmembranevoltage differences, pHluorin-based reporters of synaptic vesiclefusion, and genetically encoded calcium indicators. In a preferredembodiment, a genetically encoded voltage indicator is used. Geneticallyencoded voltage indicators that may be used or modified for use withmethods of the invention include FlaSh (Siegel, 1997, A geneticallyencoded optical probe of membrane voltage. Neuron 19:735-741); SPARC(Ataka, 2002, A genetically targetable fluorescent probe of channelgating with rapid kinetics, Biophys J 82:509-516); and VSFP1 (Sakai etal., 2001, Design and characterization of a DNA encoded,voltage-sensitive fluorescent protein, Euro J Neuroscience13:2314-2318). A genetically encoded voltage indicator based on thepaddle domain of a voltage-gated phosphatase is CiVSP (Murata et al.,2005, Phosphoinositide phosphatase activity coupled to an intrinsicvoltage sensor, Nature 435:1239-1243). Another indicator is the hybridhVOS indicator (Chanda et al., 2005, A hybrid approach to measuringelectrical activity in genetically specified neurons, Nat Neuroscience8:1619-1626), which transduces the voltage dependent migration ofdipicrylamine (DPA) through the membrane leaflet to “dark FRET”(fluorescence resonance energy transfer) with a membrane-targeted GFP.Methods of the invention may use a genetically encoded voltage indicatorin which a fluorescent moiety is inserted in the voltage sensing domain.For example, in Accelerated Sensor of Action Potentials 1 (ASAP1), acircularly permuted green fluorescent protein is inserted in anextracellular loop of a voltage-sensing domain, rendering fluorescenceresponsive to membrane potential. In some embodiments, ASAP1 is used asa reporter. ASAP1 is described in St-Pierre et al., 2014, High-fidelityoptical reporting of neuronal electrical activity with an ultrafastfluorescent voltage sensor, Nature Neuroscience 17(6):884-889.

In certain embodiments, an optical reporter of electrical activity in acell is provided by a microbial rhodopsin or a modified microbialrhodopsin. A typical microbial rhodopsin is a light-driven proton pumpstructured as an integral membrane protein belonging to the family ofarchaeal rhodopsins. Archaeal rhodopsins are characterized by seventransmembrane helices with a retinal chromophore buried therein, theretinal chromophore being covalently bound to conserved lysine residuein one of the helices via a Schiff base. See Neutze et al., 2002,Bacteriorhodopsin: a high-resolution structural view of vectorial protontransport, Biochimica et Biophysica Acta 1565:144-167; Beja et al.,2001, Proteorhodopsin phototrophy in the ocean, Nature 411:786-789. Theinvention includes the insight that microbial rhodopsins or modifiedmicrobial rhodopsins that have reduced ion pumping activity—compared tothe natural microbial rhodopsin protein from which they are derived—canbe used as an optically detectable sensor to sense voltage acrossmembranous structures, such as in cells and sub-cellular organelles whenthey are present in the lipid bilayer membrane. That is, the microbialrhodopsin proteins and the modified microbial rhodopsin proteins can beused as optical reporters to measure changes in membrane potential of acell, including prokaryotic and eukaryotic cells. The optical reportersdescribed herein are not constrained by the need for electrodes andpermit electrophysiological studies to be performed in e.g., subcellularcompartments (e.g., mitochondria) or in small cells (e.g., bacteria).The optical reporters described herein can be used in methods for drugscreening, in research settings, and in in vivo imaging systems.

The retinal chromophore imbues microbial rhodopsins with unusual opticalproperties. The linear and nonlinear responses of the retinal are highlysensitive to interactions with the protein host: small changes in theelectrostatic environment can lead to large changes in absorptionspectrum. These electro-optical couplings provide the basis for voltagesensitivity in microbial rhodopsins.

Some of the optical reporters described herein are natural proteinswithout modifications and are used in cells that do not normally expressthe microbial rhodopsin transfected to the cell, such as eukaryoticcells. For example, as shown in the examples, the wild typeArchaerhodopsin 3 can be used in neural cells to specifically detectmembrane potential and changes thereto.

Some of the microbial rhodopsins are derived from a microbial rhodopsinprotein by modification of the protein to reduce or inhibitlight-induced ion pumping of the rhodopsin protein. Such modificationspermit the modified microbial rhodopsin proteins to sense voltagewithout altering the membrane potential of the cell with its native ionpumping activity. Other mutations impart other advantageous propertiesto microbial rhodopsin voltage sensors, including increased fluorescencebrightness, improved photostability, tuning of the sensitivity anddynamic range of the voltage response, increased response speed, andtuning of the absorption and emission spectra.

Provided herein are illustrative exemplary optical voltage reporters anddirections for making and using such sensors. Other sensors that work ina similar manner as optical reporters can be prepared and used based onthe description and the examples provided herein.

Exemplary microbial rhodopsins include: green-absorbing proteorhodopsin(GPR, Gen Bank #AF349983), a light-driven proton pump found in marinebacteria; blue absorbing proteorhodopsin (BPR, GenBank # AF349981), alight-driven proton pump found in marine bacteria; Natronomonaspharaonis sensory rhodopsin II (NpSRII, GenBank # Z35086.1), alight-activated signaling protein found in the halophilic bacterium N.pharaonis; bacteriorhodopsin (BR, GenBank # NC_(—)010364.1), alight-driven proton pump found in Halobacterium salinarum;Archaerhodopsin 3 (Arch3, GenBank # P96787), a light-driven proton pumpfound in Halobacterium sodomense; variants of the foregoing; and othersdiscussed herein. Additional rhodopsions that can be mutated asindicated in the methods of the invention include fungal opsin relatedprotein (Mac, GenBank # AAG01180); Cruxrhodopsin (Crux, GenBank #BAA06678); Algal bacteriorhodopsin (Ace, GenBank # AAY82897);Archaerhodopsin 1 (Arch 1, GenBank # P69051); Archaerhodopsin 2 (Arch 2,GenBank # P29563); and Archaerhodopsin 4 (Arch 4, GenBank # AAG42454).Some of the foregoing are pointed to by Genbank number. However, arhodopsin may vary from a sequence in GenBank. Based on the descriptionof the motif described herein, a skilled artisan will easily be able tomake homologous mutations in microbial rhodopsin genes to achieve thedescribed or desired functions, e.g. reduction in the pumping activityof the microbial rhodopsin in question.

In one embodiment, the green-absorbing proteorhodopsin (GPR) is used asa starting molecule to provide an optical reporter. This molecule isselected for its relatively red-shifted absorption spectrum and its easeof expression in heterologous hosts such as E. coli. In anotherembodiment, the blue-absorbing proteorhodopsin (BPR) is used as anoptical reporter of voltage. Microbial rhodopsins are sensitive toquantities other than voltage. Mutants of GPR and BPR, as describedherein, are also sensitive to intracellular pH. It is also contemplatedthat mutants of halorhodopsin may be sensitive to local chlorideconcentration. GPR has seven spectroscopically distinguishable statesthat it passes through in its photocycle. In principle the transitionbetween any pair of states is sensitive to membrane potential. In oneembodiment, the acid-base equilibrium of the Schiff base is chosen asthe wavelength-shifting transition, hence the name of the reporter:Proteorhodopsin Optical Proton Sensor (PROPS). The absorption spectrumof wild-type GPR is known to depend sensitively on the state ofprotonation of the Schiff base. When protonated, the absorption maximumis at 545 nm, and when deprotonated the maximum is at 412 nm. When GPRabsorbs a photon, the retinal undergoes a 13-trans to cis isomerization,which causes a proton to hop from the Schiff base to nearby Asp97,leading to a shift from absorption at 545 nm to 412 nm. The PROPS designdescribed herein seeks to recapitulate this shift in response to achange in membrane potential.

Two aspects of wild-type GPR can be changed for it to serve as anoptimal voltage sensor. First, the pKa of the Schiff base can be shiftedfrom its wild-type value of 12 to a value close to the ambient pH. WhenpKa approximately equals pH, the state of protonation becomes maximallysensitive to the membrane potential. Second, the endogenouscharge-pumping capability can be eliminated so the reporter does notperturb the quantity under study. However, in some situations, a wildtype microbial rhodopsin can be used, such as Arch 3 WT, which functionsin neurons to measure membrane potential as shown in our examples.

In one embodiment, a single point mutation induces both changes in GPR.Mutating Asp97 to Asn eliminates a negative charge near the Schiff base,and destabilizes the proton on the Schiff base. The pKa shifts fromabout 12 to 9.8. In wild-type GPR, Asp97 also serves as the protonacceptor in the first step of the photocycle, so removing this aminoacid eliminates proton pumping. This mutant of GPR is referred to hereinas PROPS. Similarly, in an analogous voltage sensor derived from BPR,the homologous mutation Asp99 to Asn lowers the pKa of the Schiff baseand eliminates the proton-pumping photocycle. Thus, in one embodimentthe optical reporter is derived from BPR in which the amino acid residueAsp99 is mutated to Asn.

In GPR, additional mutations shift the pKa closer to the physiologicalvalue of 7.4. In particular, mutations Glu108 to Gln and Glu142 to Glnindividually or in combination lead to decreases in the pKa and tofurther increases in the sensitivity to voltage. Many mutations otherthan those discussed herein may lead to additional changes in the pKaand improvements in the optical properties of PROPS and are contemplatedherein.

The invention provides reporters based on rhodopsins with introducedmutations. For example, mutations that eliminate pumping in microbialrhodopsins in the present invention generally comprise mutations to theSchiff base counterion; a carboxylic amino acid (Asp or Glu) conservedon the third transmembrane helix (helix C) of the rhodopsin proteins.Mutations to the carboxylic residue directly affect the protonconduction pathway, eliminating proton pumping (e.g., Asp to Asn, Gln,or His mutation, or Glu to Asn Gln, or His mutation). Mutating theproton acceptor aspartic acid adjacent the Schiff base to asparaginesuppresses proton pumping. Thus, in some embodiments, the mutations areselected from the group consisting of: D97N (green-absorbingproteorhodopsin), D95N (Archaerhodopsin 3), D99N (blue-absorbingproteorhodopsin), D75N (sensory rhodopsin II), and D85N(bacteriorhodopsin). In other embodiments, residues that can be mutatedto inhibit pumping include (using bacteriorhodopsin numbering) D96,Y199, and R82, and their homologues in other microbial rhodopsins. Inanother embodiment, residue D95 can be mutated in Archaerhodopsin toinhibit proton pumping (e.g., D95N). Residues near the binding pocketcan be mutated singly or in combination to tune the spectra to a desiredabsorption and emission wavelength. In bacteriorhodopsin these residuesinclude, but are not limited to, L92, W86, W182, D212, 1119, and M145.Homologous residues may be mutated in other microbial rhodopsins. Thus,in some embodiments, the mutation to modify the microbial rhodopsinprotein is performed at a residue selected from the group consisting ofL92, W86, W182, D212, 1119, M145. Mutations can shift the dynamic rangeof voltage sensitivity into a desired band by shifting the distributionof charge in the vicinity of the Schiff base, and thereby changing thevoltage needed to add or remove a proton from this group.Voltage-shifting mutations in green-absorbing proteorhodopsin include,but are not limited to, E108Q, E142Q, L217D, either singly or incombination using green-absorbing proteorhodopsin locations as anexample, or a homologous residue in another rhodopsin. In oneembodiment, a D95N mutation is introduced into Archaerhodopsin 3 toadjust the pKa of the Schiff base towards a neutral pH. Additionally oralternatively, mutations can enhance brightness, photostability, orboth. Residues which, when mutated, may restrict the binding pocket toincrease fluorescence include (using bacteriorhodopsin numbering) Y199,Y57, P49, V213, and V48.

Optical reporters that may be suitable for use with the inventioninclude those that use the endogenous fluorescence of the microbialrhodopsin protein Archaerhodopsin 3 (Arch) from Halorubum sodomense.Arch resolves action potentials with high signal-to-noise (SNR) and lowphoto-toxicity.

FIG. 4 shows genetically encoded fluorescent voltage indicatorsclassified according to their sensitivity and speed—the two keyparameters that determine the performance of an indicator. The inventionprovides reporters such as Proteorhodopsin Optical Proton Sensor(PROPS), Arch 3 WT, and Arch 3 D95N, shown on the upper right. PROPSfunctions in bacteria, while Arch 3 WT and Arch 3 D95N function inmammalian cells. Such microbial rhodopsin-based voltage indicators arefaster and far more sensitive than other indicators.

The invention may use optical reporters that include fluorescent voltageindicating proteins such as VSFP 2.3 (Knopfel et al., 2010, J Neurosci30:14998-15004), which exhibits a response time of 78 ms and f (wheref=(delta F/F per 100 mV)) of 9.5%. VSFP 2.4 (Ibid.) has a 72 ms responsetime and f of 8.9%. VSFP 3.1 (Lundby et al., 2008, PLoSOne 3:2514) has aresponse time of 1-20 ms and a F of 3%. Mermaid is a molecule describedin Perron et al., 2009, Front Mol Neurosci 2:1-8 with a response time of76 and a F of 9.2%. SPARC (Ataka & Pieribone, 2002, Biophys J82:509-516) response time 0.8 ms and F 0.5%. Flash (Siegel, 1997, Neuron19:735-741) has response time 2.8-85 ms and f of 5.1%. Arch 3 WT has aresponse time of <0.5 ms and f of 66%. Arch D95N has a response time of41 ms and f of 100%.

Optical recording of action potentials were made in a single rathippocampal neuron.

FIG. 5 shows electrically recorded membrane potential of a neuronexpressing Arch WT, subjected to pulses of current injection and laserillumination (I=1800 W/cm2, λ=640 nm). Illumination generated sufficientphotocurrent to suppress action potentials when the cell was nearthreshold. Grey bars indicate laser illumination.

FIG. 6 shows electrically recorded membrane potential of a neuronexpressing Arch D95N, showing no effect of illumination on spiking orresting potential. Experiments have shown a neuron expressing Arch D95N,showing Arch D95N fluorescence (shows in cyan in the experiment), andregions of voltage-dependent fluorescence (shown in red in theexperiment).

FIG. 7 shows whole-cell membrane potential determined via electricalrecording (bottom, voltage line) and weighted ArchD95N fluorescence(top, fluorescence line) during a single-trial recording of a train ofaction potentials. The data represents a single trial, in which spikingwas induced by injection of a current pulse. The fluorescence showsclear bursts accompanying individual action potentials. This experimentis the first robust measurement of action potentials in a singlemammalian neuron using a genetically encoded voltage indicator.

FIG. 8 shows optical recordings of action potentials in a single HL-1mouse cardiomyocyte expressing Arch 3 D95N-eGFP. Action potentials wererecorded for up to 1000 s, with no signs of phototoxicity. Thisexperiment shows quantitative measurement of cardiac action potentialswith a genetically encoded voltage indicator. An overlay showsfluorescence of Arch D95N and GFP in a Arch D95N-GFP fusion. FIG. 8shows a comparison of the action potential determined from patch clamprecording (dashed line) and fluorescence (solid line). FIG. 9 showsoptical recordings of the action potentials in a single HL-1 cell overtime.

Arch and the above-mentioned variants target eukaryotic membranes andcan image single action potentials and subthreshold depolarization incultured mammalian neurons. See Kralj et al, 2012, Optical recording ofaction potentials in mammalian neurons using a microbial rhodopsin, NatMethods 9:90-95. See Hochbaum et al., All-optical electrophysiology inmammalian neurons using engineered microbial rhodopsins, Nature Methods,published online Jun. 22, 2014. Thus Arch and variants of Arch mayprovide good optical reporters of neural activity according toembodiments of the invention.

The invention provides optical reporters based on Archaerhodopsins thatfunction in mammalian cells, including human stem cell-derived neuronsand cardiomyocytes. These proteins indicate electrical dynamics withsub-millisecond temporal resolution and sub-micron spatial resolutionand may be used in non-contact, high-throughput, and high-contentstudies of electrical dynamics in cells and tissues using opticalmeasurement of membrane potential. These reporters are broadly useful,particularly in eukaryotic, such as mammalian, including human cells.

The invention includes reporters based on Archaerhodopsin 3 (Arch 3) andits homologues. Arch 3 is Archaerhodopsin from H. sodomense and it isknown as a genetically-encoded reagent for high-performanceyellow/green-light neural silencing. Gene sequence at GenBank:GU045593.1 (synthetic construct Arch 3 gene, complete cds. SubmittedSep. 28, 2009). These proteins localize to the plasma membrane ineukaryotic cells and show voltage-dependent fluorescence.

Exemplary sequences that can be used to generate virus constructs withArch 3 include a lentivirus backbone with promoters such as CamKII(excitatory neuron specific); hSynapsin (pan neuronal); CAG enhancer(pan cellular); CMV (pan cellular); Ubiquitin (pan cellular); others; ora combination thereof.

The invention may use optical reporters that include fluorescent voltageindicating proteins such as VSFP 2.3 (Knopfel et al., 2010, Toward thesecond generation of optogenetic tools, J Neurosci 30:14998-15004),which exhibits a response time of 78 ms and f (where f=(delta F/F per100 mV)) of 9.5%. VSFP 2.4 (Ibid.) has a 72 ms response time and f of8.9%. VSFP 3.1 (Lundby et al., 2008, Engineering of a geneticallyencodable fluorescent voltage sensor exploiting fast Ci-VSPvoltage-sensing movements, PLoSOne 3:2514) has a response time of 1-20ms and a f of 3%. Mermaid is a molecule described in Perron et al.,2009, Second and third generation voltage-sensitive fluorescent proteinsfor monitoring membrane potential, Front Mol Neurosci 2:1-8 with aresponse time of 76 and a F of 9.2%. SPARC (Ataka & Pieribone, 2002,Biophys J 82:509-516) response time 0.8 ms and F 0.5%. Flash (Siegel,1997, Neuron 19:735-741) has response time 2.8-85 ms and f of 5.1%. Arch3 WT has a response time of 0.6 ms and f of 32%.

Fluorescence recordings may be acquired on an epifluorescencemicroscope, described in Kralj et al., 2012, Optical recording of actionpotentials in mammalian neurons using a microbial rhodopsin, Nat.Methods 9:90-95.

Optical reporters of the invention show high sensitivity. In mammaliancells, optical reporters show about 3-fold increase in fluorescencebetween −150 mV and +150 mV. The response is linear over most of thisrange. Membrane voltage can be measured with a precision of <1 mV in a 1s interval. Reporters of the invention show high speed. Arch 3 WT shows90% of its step response in 0.6 ms. A neuronal action potential lastsapproximately 1 ms, so the speeds of Arch indicators meet the benchmarkfor imaging electrical activity of neurons. Arch 3 WT retains thephoto-induced proton-pumping, so illumination slightly hyperpolarizesthe cell. Reporters of the invention show high photo-stability and arecomparable to GFP in the number of fluorescence photons produced priorto photobleaching. The reporters may also show far red spectrum. Thevoltage-indicating protein reporters, sometimes referred to asgenetically encoded voltage indicators (GEVIs), may be excited with alaser at wavelengths between 590-640 nm, and the emission is in the nearinfrared, peaked at 710 nm. The emission is farther to the red than anyexisting fluorescent protein. These wavelengths coincide with lowcellular auto-fluorescence and good transmission through tissue. Thisfeature makes these proteins particularly useful in optical measurementsof action potentials as the spectrum facilitates imaging with highsignal-to-noise ratio, as well as multi-spectral imaging in combinationwith other fluorescent probes.

The reporters can be targeted to specific locations or cell typesincluding primary neuronal cultures, cardiomyocytes (HL-1 and humaniPSC-derived), HEK cells, and Gram positive and Gram negative bacteriaas well as to the endoplasmic reticulum, and to mitochondria. Theconstructs are useful also for in vivo imaging in C. elegans, zebrafish,mice, and rats. Using promoters specific to a particular cell type,time, or both, membrane potential may be imaged in any opticallyaccessible cell type or organelle in a living organism. A reporter mayinclude at least three elements: a promoter, a microbial rhodopsinvoltage sensor, one or more targeting motifs, and an optional accessoryfluorescent protein. Some non-limiting examples for each of theseelements are rhodopsins are given above. Exemplary promoters includeCMV, 14× UAS-E1b, HuC, ara, and lac. Exemplary targeting motifs includeSS (beta-2nAChR) SS (PPL), ER export motif, TS from Kir2.1, and MS.Exemplary fluorescent proteins include Venus, EYFP, and TagRFP.

In one embodiment, at least one or more rhodopsin, promoter, targetingmotif, and fluorescent protein is selected to create an optical voltagesensor with the desired properties. In some embodiments, methods andcompositions for voltage sensing as described herein involvesselecting: 1) a microbial rhodopsin protein, 2) one or more mutations toimbue the protein with sensitivity to voltage or to other quantities ofinterest and to eliminate light-driven charge pumping, 3) codon usageappropriate to the host species, 4) a promoter and targeting sequencesto express the protein in cell types of interest and to target theprotein to the sub-cellular structure of interest, 5) an optional fusionwith a conventional fluorescent protein to provide ratiometric imaging,6) a chromophore to insert into the microbial rhodopsin, and 7) anoptical imaging scheme.

In one embodiment, the voltage sensor is selected from a microbialrhodopsin protein (wild-type or mutant) that provides a voltage-inducedshift in its absorption or fluorescence. The starting sequences fromwhich these constructs can be engineered include, but are not limitedto, the rhodopsins and mutations discussed herein that can be made tothe gene to enhance the performance of the protein product.

4b. Optogenetic Actuator

In a preferred embodiment, the cells are transformed with an opticalvoltage actuator. This can occur, for example, simultaneously withtransformation with the vector comprising the optogenetic reporter. Thefar-red excitation spectrum of certain Arch-based reporters suggeststhat they may be paired with a blue light-activated channelrhodopsin toachieve all-optical electrophysiology. For spatially precise opticalexcitation, the channelrhodopsin should carry current densitiessufficient to induce action potentials (APs) when only a subsection of acell is excited. Preferably, light used for imaging the reporter shouldnot activate the actuator, and light used for activating the actuatorshould not confound the fluorescence signal of the reporter. Thus in apreferred embodiment, an optical actuator and an optical reporter arespectrally orthogonal to avoid crosstalk and allow for simultaneous use.Spectrally orthogonal systems are discussed in Carlson and Campbell,2013, Circular permutated red fluorescent proteins and calcium ionindicators based on mCherry, Protein Eng Des Sel 26(12):763-772.

Preferably, a genetically-encoded optogenetic actuator is used. Oneactuator is channelrhodopsin2 H134R, an optogenetic actuator describedin Nagel, G. et al., 2005, Light activation of channelrhodopsin-2 inexcitable cells of Caenorhabditis elegans triggers rapid behavioralresponses, Curr Biol 15:2279-2284.

A screen of plant genomes has identified an optogenetic actuator,Scherffelia dubia ChR (sdChR), derived from a fresh-water green algafirst isolated from a small pond in Essex, England. See Klapoetke etal., 2014, Independent optical excitation of distinct neuralpopulations, Nat Meth Advance Online Publication 1-14; see alsoMelkonian & Preisig, 1986, A light and electron microscopic study ofScherffelia dubia, a new member of the scaly green flagellates(Prasinophyceae). Nord. J. Bot. 6:235-256, both incorporated byreference. SdChR may offer good sensitivity and a blue action spectrum.

An improved version of sdChR dubbed CheRiff may be used as an opticalactuator. The gene for Scherffelia dubia Channelrhodopsin (sdChR)(selected from a screen of channelrhodopsins for its blue excitationpeak (474 nm) and its large photocurrent relative to ChR2) issynthesized with mouse codon optimization, a trafficking sequence fromKir2.1 is added to improve trafficking, and the mutation E154A isintroduced. CheRiff exhibits significantly decreased crosstalk from redillumination (to 10.5±2.8 pA) allowing its use in cells along withoptogenetic reporters described herein. CheRiff shows good expressionand membrane trafficking in cultured rat hippocampal neurons. Themaximum photocurrent under saturating illumination (488 nm, 500 mW/cm²)is 2.0±0.1 nA (n=10 cells), approximately 2-fold larger than the peakphotocurrents of ChR2 H134R or ChIEF (Lin et al., 2009, Characterizationof engineered channelrhodopsin variants with improved properties andkinetics, Biophys J 96:1803-1814). In neurons expressing CheRiff,whole-cell illumination at only 22±10 mW/cm² induces a photocurrent of 1nA. Compared to ChR2 H134R and to ChIEF under standard channelrhodopsinillumination conditions (488 nm, 500 mW/cm²). At 23° C., CheRiff reachespeak photocurrent in 4.5±0.3 ms (n=10 cells). After a 5 ms illuminationpulse, the channel closing time constant was comparable between CheRiffand ChIEF (16±0.8 ms, n=9 cells, and 15±2 ms, n=6 cells, respectively,p=0.94), and faster than ChR2 H134R (25±4 ms, n=6 cells, p<0.05). Undercontinuous illumination CheRiff partially desensitizes with a timeconstant of 400 ms, reaching a steady-state current of 1.3±0.08 nA (n=10cells). Illumination of neurons expressing CheRiff induces trains of APswith high reliability and high repetition-rate.

When testing for optical crosstalk between Arch-based reporters andCheRiff in cultured neurons, illumination sufficient to inducehigh-frequency trains of APs (488 nm, 140 mW/cm²) perturbed fluorescenceof reporters by <1%. Illumination with high intensity red light (640 nm,900 W/cm²) induced an inward photocurrent through CheRiff of 14.3±3.1pA, which depolarized neurons by 3.1±0.2 mV (n=5 cells). ChIEF and ChR2H134R generated similar red light photocurrents and depolarizations. Formost applications this level of optical crosstalk is acceptable.

4c. Vectors for Expression of Optogenetic Systems

The optogenetic reporters and actuators may be delivered in constructsdescribed here as Optopatch constructs delivered through the use of anexpression vector. Optopatch may be taken to refer to systems thatperform functions traditionally associated with patch clamps, but via anoptical input, readout, or both as provided for by, for example, anoptical reporter or actuator. An Optopatch construct may include abicistronic vector for co-expression of CheRiff-eGFP and a reporter(e.g., a suitable Arch-based reporter such as Arch D95N). The reporterand CheRiff constructs may be delivered separately, or a bicistronicexpression vector may be used to obtain a uniform ratio of actuator toreporter expression levels.

The genetically encoded reporter, actuator, or both may be delivered byany suitable expression vector using methods known in the art. Anexpression vector is a specialized vector that contains the necessaryregulatory regions needed for expression of a gene of interest in a hostcell. Examples of vectors include plasmids (e.g. pBADTOPO, pCI-Neo,pcDNA3.0), cosmids, and viruses (such as a lentivirus, anadeno-associated virus, or a baculovirus). In some embodiments the geneof interest is operably linked to another sequence in the vector. Insome embodiments, it is preferred that the viral vectors are replicationdefective, which can be achieved for example by removing all viralnucleic acids that encode for replication. A replication defective viralvector will still retain its infective properties and enters the cellsin a similar manner as a replicating vector, however once admitted tothe cell a replication defective viral vector does not reproduce ormultiply. The term “operably linked” means that the regulatory sequencesnecessary for expression of the coding sequence are placed in the DNAmolecule in the appropriate positions relative to the coding sequence soas to effect expression of the coding sequence. This same definition issometimes applied to the arrangement of coding sequences andtranscription control elements (e.g. promoters, enhancers, andtermination elements) in an expression vector.

Many viral vectors or virus-associated vectors are known in the art.Such vectors can be used as carriers of a nucleic acid construct intothe cell. Constructs may be integrated and packaged intonon-replicating, defective viral genomes like Adenovirus,Adeno-associated virus (AAV), serotypes of AAV that include AAV1-AAV9,or Herpes simplex virus (HSV) or others, including retroviral andlentiviral vectors, for infection or transduction into cells. The vectormay or may not be incorporated into the cell's genome. The constructsmay include viral sequences for transfection, if desired. Alternatively,the construct may be incorporated into vectors capable of episomalreplication, such as an Epstein Barr virus (EPV or EBV) vector. Theinserted material of the vectors described herein may be operativelylinked to an expression control sequence when the expression controlsequence controls and regulates the transcription and translation ofthat polynucleotide sequence. In some examples, transcription of aninserted material is under the control of a promoter sequence (or othertranscriptional regulatory sequence) which controls the expression ofthe recombinant gene. In some embodiments, a recombinant cell containingan inducible promoter is used and exposed to a regulatory agent orstimulus by externally applying the agent or stimulus to the cell ororganism by exposure to the appropriate environmental condition or theoperative pathogen. Inducible promoters initiate transcription only inthe presence of a regulatory agent or stimulus. Examples of induciblepromoters include the tetracycline response element and promotersderived from the beta-interferon gene, heat shock gene, metallothioneingene or any obtainable from steroid hormone-responsive genes. Induciblepromoters which may be used in performing the methods of the presentinvention include those regulated by hormones and hormone analogs suchas progesterone, ecdysone and glucocorticoids as well as promoters whichare regulated by tetracycline, heat shock, heavy metal ions, interferon,and lactose operon activating compounds. See Gingrich and Roder, 1998,Inducible gene expression in the nervous system of transgenic mice, AnnuRev Neurosci 21:377-405. Tissue specific expression has been wellcharacterized in the field of gene expression and tissue specific andinducible promoters are well known in the art. These promoters are usedto regulate the expression of the foreign gene after it has beenintroduced into the target cell. In certain embodiments, a cell-typespecific promoter or a tissue-specific promoter is used. A cell-typespecific promoter may include a leaky cell-type specific promoter, whichregulates expression of a selected nucleic acid primarily in one celltype, but cause expression in other cells as well. For expression of anexogenous gene specifically in neuronal cells, a neuron-specific enolasepromoter can be used. See Forss-Petter et al., 1990, Transgenic miceexpressing beta-galactosidase in mature neurons under neuron-specificenolase promoter control, Neuron 5(2):187-197.

Suitable delivery methods include viral and non-viral vectors, as wellas biological or chemical methods of transfection. The methods can yieldeither stable or transient gene expression in the system used. In someembodiments, a viral vector such as an (i) adenovirus, (ii)adeno-associated virus, (iii) retrovirus, (iv) lentivirus, or (v) otheris used.

(i) Adenovirus

Adenoviruses are double stranded, non-enveloped and icosahedral virusescontaining a 36 kb viral genome (Kojaoghlanian et al., 2003, The impactof adenovirus infection on the immunocompromised host, Rev Med Virol13:155-171). Their genes are divided into early (E1A, E1B, E2, E3, E4),delayed (IX, IVa2) and major late (L1, L2, L3, L4, L5) genes dependingon whether their expression occurs before or after DNA replication. Morethan 51 human adenovirus serotypes have been described which can infectand replicate in a wide range of organs. These viruses have been used togenerate a series of vectors for gene transfer cellular engineering. Theinitial generation of adenovirus vectors were produced by deleting theE1 gene (required for viral replication) generating a vector with a 4 kbcloning capacity. An additional deletion of E3 (responsible for hostimmune response) allowed an 8 kb cloning capacity (Bett et al., 1994, Anefficient and flexible system for construction of adenovirus vectorswith insertions or deletions in early regions 1 and 3, PNAS91:8802-8806; Danthinne and Imperiale, 2000, Production of firstgeneration adenovirus vector, a review, Gene Ther 7:1707-1714). Thesecond generation of vectors was produced by deleting the E2 region(required for viral replication) and/or the E4 region (participating ininhibition of host cell apoptosis) in conjunction with E1 or E3deletions. The resultant vectors have a cloning capacity of 10-13 kb(Armentano et al., 1995, Characterization of an adenovirus gene transfervector containing an E4 deletion, Hum Gen Ther 6(10):1343-1353). Thethird “gutted” generation of vectors was produced by deletion of theentire viral sequence with the exception of the inverted terminalrepeats (ITRs) and the cis acting packaging signals. These vectors havea cloning capacity of 25 kb (Kochanek et al., 2001, High-capacity“gutless” adenoviral vectors, Curr Op Mol Ther 3:454-463) and haveretained their high transfection efficiency both in quiescent anddividing cells.

Importantly, the adenovirus vectors do not normally integrate into thegenome of the host cell, but they have shown efficacy for transient genedelivery into adult stem cells. These vectors have a series ofadvantages and disadvantages. An important advantage is that they can beamplified at high titers and can infect a wide range of cells. Thevectors are generally easy to handle due to their stability in variousstoring conditions. Adenovirus type 5 (Ad5) has been successfully usedin delivering genes in human and mouse stem cells and withoutintegration generally provides transient expression.

(ii) Adeno-Associated Virus

Adeno-Associated viruses (AAV) are ubiquitous, noncytopathic,replication-incompetent members of ssDNA animal virus of parvoviridaefamily (Gao et al., 2005, New recombinant serotypes of AAV vectors, CurrGene Ther 5 (3):285-97). AAV is a small icosahedral virus with a 4.7 kbgenome. These viruses have a characteristic termini consisting ofpalindromic repeats that fold into a hairpin. They replicate with thehelp of helper virus, which are usually one of the many serotypes ofadenovirus. In the absence of helper virus they integrate into the humangenome at a specific locus (AAVS1) on chromosome 19 and persist inlatent form until helper virus infection occurs. AAV can transduce celltypes from different species including mouse, rat and monkey. Theseviruses are similar to adenoviruses in that they are able to infect awide range of dividing and non-dividing cells. Unlike adenovirus, theyhave the ability to integrate into the host genome at a specific site inthe human genome.

In some embodiments the viral vector is an adeno-associated virus (AAV)vector. AAV can infect both dividing and non-dividing cells and mayincorporate its genome into that of the host cell. One suitable viralvector uses recombinant adeno-associated virus (rAAV), which is widelyused for gene delivery in the CNS. In certain embodiments, the vectormay use AAV serotype 9 (AAV9). See Bell et al., 2011, The AAV9 receptorand its modification to improve in vivo lung gene transfer in mice, JClin Invest 121(6):2427-2435; and Cearley & Wolfe, 2006, Transductioncharacteristics of adeno-associated virus vectors expressing capserotypes 7, 8, 9, and Rh10 in the mouse brain, Mol Ther 13:528-537; andFoust et al., 2009, Intravascular AAV9 preferentially targets neonatalneurons and adult astrocytes, Nat Biotechnol 27:59-65.

(ii) Retroviruses

Retroviral genomes consist of two identical copies of single strandedpositive sense RNAs, 7-10 kb in length coding for three genes; gag, poland env, flanked by long terminal repeats (LTR) (Yu & Schaffer, 2006,Engineering retroviral and lentiviral vectors by selection of a novelpeptide insertion library for enhanced purification, J. Virol.80:3285-3292). The gag gene encodes the core protein capsid containingmatrix and nucleocapsid elements that are cleavage products of the gagprecursor protein. The pol gene codes for the viral protease, reversetranscriptase and integrase enzymes derived from gag-pol precursor gene.The env gene encodes the envelop glycoprotein which mediates viralentry. An important feature of the retroviral genome is the presence ofLTRs at each end of the genome. These sequences facilitate theinitiation of viral DNA synthesis, moderate integration of the proviralDNA into the host genome, and act as promoters in regulation of viralgene transcription. Retroviruses are subdivided into three generalgroups: the oncoretroviruses (Maloney Murine Leukenmia Virus, MoMLV),the lentiviruses (HIV), and the spumaviruses (foamy virus). Retroviralbased vectors are the most commonly used integrating vectors for genetherapy. These vectors generally have a cloning capacity ofapproximately 8 kb and are generated by a complete deletion of the viralsequence with the exception of the LTRs and the cis acting packagingsignals.

(ii) Lentivirus

Lentiviruses are members of Retroviridae family of viruses (Scherr etal., 2002, Gene transfer into hematopoietic stem cells using lentiviralvectors, Curr Gene Ther. 2(1):45-55). They have a more complex genomeand replication cycle as compared to the oncoretroviruses (Beyer et al.,2002, Oncoretrovirus and lentivirus vectors pseudotyped with lymphocyticchoriomeningitis virus glycoprotein: generation, concentration, andbroad host range, J. Virol 76:1488-1495). They differ from simplerretroviruses in that they possess additional regulatory genes andelements, such as the tat gene, which mediates the transactivation ofviral transcription and rev, which mediates nuclear export of un-splicedviral RNA. See also U.S. Pat. No. 5,665,577 to Sodroski, the contents ofwhich are incorporated by reference.

Lentivirus vectors are derived from the human immunodeficiency virus(HIV-1) by removing the genes necessary for viral replication renderingthe virus inert. Although they are devoid of replication genes, thevector can still efficiently integrate into the host genome allowingstable expression of the transgene. These vectors have the additionaladvantage of a low cytotoxicity and an ability to infect diverse celltypes.

Lentiviral vectors may include a eukaryotic promoter. The promoter canbe any inducible promoter, including synthetic promoters, that canfunction as a promoter in a eukaryotic cell. For example, the eukaryoticpromoter can be, but is not limited to, CamKIIa promoter, human Synapsinpromoter, ecdysone inducible promoters, E1a inducible promoters,tetracycline inducible promoters etc., as are well known in the art. Inaddition, the lentiviral vectors used herein can further comprise aselectable marker, which can comprise a promoter and a coding sequencefor a selectable trait. Nucleotide sequences encoding selectable markersare well known in the art, and include those that encode gene productsconferring resistance to antibiotics or anti-metabolites, or that supplyan auxotrophic requirement. Examples of such sequences include, but arenot limited to, those that encode thymidine kinase activity, orresistance to methotrexate, ampicillin, kanamycin, among others. Use oflentiviral vectors is discussed in Wardill et al., 2013, A neuron-basedscreening platform for optimizing genetically-encoded calciumindicators, PLoS One 8(10):e77728; Dottori, et al., Neural developmentin human embryonic stem cells-applications of lentiviral vectors, J CellBiochem 112(8):1955-62; and Diester et al., 2011, An optogenetic toolboxdesigned for primates, Nat Neurosci 14(3):387-97. When expressed under aCaMKIIα promoter in cultured rat hippocampal neurons the Optopatchconstruct exhibits high expression and good membrane trafficking of bothCheRiff and a reporter.

In certain embodiments, genetic material is delivered by a non-viralmethod. Non-viral methods include plasmid transfer, modified RNA, andthe application of targeted gene integration through the use ofintegrase or transposase technologies. Exemplary recombinase systemsinclude: cre recombinase from phage P1 (Lakso et al., 1992, Targetedoncogene activation by site-specific recombination in transgenic mice,PNAS 89:6232-6236; Orban et al., 1992, Tissue- and site-specific DNArecombination in transgenic mice, PNAS 89:6861-6865), FLP (flippase)from yeast 2 micron plasmid (Dymecki, 1998, Using Flp-recombinase tocharacterize expansion of Wnt1-expressing neural progenitors in themouse, Dev Biol 201:57-65), and an integrase isolated from streptomysesphage I C31 (Groth et al., 2000, A phage integrase directs efficientsite-specific integration in human cells, PNAS 97(11):5995-6000). Eachof these recombinases recognize specific target integration sites. Creand FLP recombinase catalyze integration at a 34 bp palindromic sequencecalled lox P (locus for crossover) and FRT (FLP recombinase target)respectively. Phage integrase catalyzes site-specific, unidirectionalrecombination between two short att recognition sites in mammaliangenomes. Recombination results in integration when the att sites arepresent on two different DNA molecules and deletion or inversion whenthe att sites are on the same molecule. It has been found to function intissue culture cells (in vitro) as well as in mice (in vivo).

The Sleeping Beauty (SB) transposon is comprised of two invertedterminal repeats of 340 base pairs each (Izsvak et al., 2000, SleepingBeauty, a wide host-range transposon vector for genetic transformationin vertebrates, J Mol Biol 302(1):93-102). This system directs theprecise transfer of specific constructs from a donor plasmid into amammalian chromosome. The excision and integration of the transposonfrom a plasmid vector into a chromosomal site is mediated by the SBtransposase, which can be delivered to cells as either in a cis or transmanner (Kaminski et al., 2002, Design of a nonviral vector forsite-selective, efficient integration into the human genome, FASEB J6:1242-1247). A gene in a chromosomally integrated transposon can beexpressed over the lifetime of a cell. SB transposons integrate randomlyat TA-dinucleotide base pairs although the flanking sequences caninfluence integration.

In certain embodiments, methods of the invention use a Cre-dependentexpression system. Cre-dependent expression includes Cre-Loxrecombination, a site-specific recombinase technology that uses theenzyme Cre recombinase, which recombines a pair of short targetsequences called the Lox sequences. This system can be implementedwithout inserting any extra supporting proteins or sequences. The Creenzyme and the original Lox site called the LoxP sequence are derivedfrom bacteriophage P1. Bacteriophage P1 uses Cre-lox recombination tocircularize and replicate its genomic DNA. This recombination strategyis employed in Cre-Lox technology for genome manipulation, whichrequires only the Cre recombinase and LoxP sites. Sauer & Henderson,1988, Site-specific DNA recombination in mammalian cells by the Crerecombinase of bacteriophage P1, PNAS 85:5166-70 and Sternberg &Hamilton, 1981, Bacteriophage P1 site-specific recombination. I.Recombination between LoxP sites, J Mol Biol 150:467-86. Methods may usea Cre recombinase-dependent viral vector for targeting tools such aschannelrhodopsin-2 (ChR2) to specific neurons with expression levelssufficient to permit reliable photostimulation. Optogenetic tools suchas ChR2 tagged with a fluorescent protein such as mCherry (e.g.,ChR2mCherry) or any other of the tools discussed herein are thusdelivered to the cell or cells for use in characterizing those cells.

The delivery vector may include Cre and Lox. The vector may furtheroptionally include a Lox-stop-Lox (LSL) cassette to prevent expressionof the transgene in the absence of Cre-mediated recombination. In thepresence of Cre recombinase, the LoxP sites recombine, and a removabletranscription termination Stop element is deleted. Removal of the stopelement may be achieved through the use of AdenoCre, which allowscontrol of the timing and location of expression. Use of the LSLcassette is discussed in Jackson, et al., 2001, Analysis of lung tumorinitiation and progression using conditional expression of oncogenicK-ras, Genes & Dev 15:3243-3248.

In certain embodiments, a construct of the invention uses a“flip-excision” switch, or FLEX switch (FLip EXicision), to achievestable transgene inversion. The FLEX switch is discussed in Schnutgen etal., 2003, A directional strategy for monitoring Cre-mediatedrecombination at the cellular level in the mouse, Nat Biotechnol21:562-565. The FLEX switch uses two pairs of heterotypic, antiparallelLoxP-type recombination sites which first undergo an inversion of thecoding sequence followed by excision of two sites, leading to one ofeach orthogonal recombination site oppositely oriented and incapable offurther recombination. A FLEX switch provides high efficiency andirreversibility. Thus in some embodiments, methods use a viral vectorcomprising rAAV-FLEX-rev-ChR2mCherry. Additionally or alternatively, avector may include FLEX and any other optogenetic tool discussed herein(e.g., rAAV-FLEX-Arch D95N, rAAV-FLEX-CheRiff). UsingrAAV-FLEX-rev-ChR2mCherry as an illustrative example, Cre-mediatedinversion of the ChR2mCherry coding sequence results in the codingsequence being in the wrong orientation (i.e., rev-ChR2mCherry) fortranscription until Cre inverts the sequence, turning on transcriptionof ChR2mCherry. FLEX switch vectors are discussed in Atasoy et al.,2009, A FLEX switch targets channelrhodopsin-2 to multiple cell typesfor imaging and long-range circuit mapping, J Neurosci 28(28):7025-7030.

Use of a viral vector such as Cre-Lox system with an optical reporter,optical actuator, or both (optionally with a FLEX switch and/or aLox-Stop-Lox cassette) for labeling and stimulation of neurons allowsfor efficient photo-stimulation with only brief exposure (1 ms) to lessthan 100 μW focused laser light or to light from an optical fiber. SuchFurther discussion may be found in Yizhar et al., 2011, Optogenetics inneural systems, Neuron 71(1):9-34; Cardin et al., 2010, Targetedoptogenetic stimulation and recording of neurons in vivo usingcell-type-specific expression of Channelrhodopsin-2, Nat Protoc5(2):247-54; Rothermel et al., 2013, Transgene expression intarget-defined neuron populations mediated by retrograde infection ithadeno-associated viral vectors, J Neurosci 33(38):195-206; and Saunderset al., 2012, Novel recombinant adeno-associated viruses for Creactivated and inactivated transgene expression in neurons, Front NeuralCircuits 6:47.

In certain embodiments, actuators, reporters, or other genetic materialmay be delivered using chemically-modified mRNA. It may be found andexploited that certain nucleotide modifications interfere withinteractions between mRNA and toll-like receptor, retinoid-induciblegene, or both. Exposure to mRNAs coding for the desired product may leadto a desired level of expression of the product in the cells. See, e.g.,Kormann et al., 2011, Expression of therapeutic proteins after deliveryof chemically modified mRNA in mice, Nat Biotech 29(2):154-7; Zangi etal., 2013, Modified mRNA directs the fate of heart progenitor cells andinduces vascular regeneration after myocardial infarction, Nat Biotech31:898-907.

It may be beneficial to culture or mature the cells after transformationwith the genetically encoded optical reporter with optional actuator. Insome embodiments, the neurons are matured for 8-10 days post infection.Using microscopy and analytical methods described herein, the cell andits action potentials may be observed. For additional discussion, seeU.S. Pub. 2013/0224756, incorporated by reference in its entirety forall purposes.

Other methods for transfection include physical methods such aselectroporation as well as methods that employ biomolecules.

Electroporation relies on the use of brief, high voltage electric pulseswhich create transient pores in the membrane by overcoming itscapacitance. One advantage of this method is that it can be utilized forboth stable and transient gene expression in most cell types. Thetechnology relies on the relatively weak nature of the hydrophobic andhydrophilic interactions in the phospholipid membrane and its ability torecover its original state after the disturbance. Once the membrane ispermeabilized, polar molecules can be delivered into the cell with highefficiency. Large charged molecules like DNA and RNA move into the cellthrough a process driven by their electrophoretic gradient.

Biomolecule-based methods include the use of protein transductiondomains (PTD). PTDs are short peptides that are transported into thecell without the use of the endocytotic pathway or protein channels. Themechanism involved in their entry is not well understood, but it canoccur even at low temperature (Derossi et al., 1996, J Biol Chem271(30):18188-93). The two most commonly used naturally occurring PTDsare the trans-activating activator of transcription domain (TAT) ofhuman immunodeficiency virus and the homeodomain of Antennapediatranscription factor. In addition to these naturally occurring PTDs,there are a number of artificial peptides that have the ability tospontaneously cross the cell membrane (Joliot and Prochiantz, 2004,Transduction peptides: from technology to physiology, Nat Cell Biol6(3):189-96). These peptides can be covalently linked to thepseudo-peptide backbone of PNA (peptide nucleic acids) to help deliverthem into the cell.

Additionally or alternatively, liposomes may be used. Liposomes aresynthetic vesicles that resemble the cell membrane. When lipid moleculesare agitated with water they spontaneously form spherical doublemembrane compartments surrounding an aqueous center forming liposomes.They can fuse with cells and allow the transfer of “packaged” materialinto the cell. Liposomes have been successfully used to deliver genes,drugs, reporter proteins and other biomolecules into cells (Felnerova etal., 2004, Liposomes and virosomes as delivery systems for antigens,nucleic acids and drugs, Curr Opin Biotech 15: 518-529). The advantageof liposomes is that they are made of natural biomolecules (lipids) andare nonimmunogenic.

Diverse hydrophilic molecules can be incorporated into them duringformation. For example, when lipids with positively charged head groupare mixed with recombinant DNA they can form lipoplexes in which thenegatively charged DNA is complexed with the positive head groups oflipid molecules. These complexes can then enter the cell through theendocytotic pathway and deliver the DNA into lysosomal compartments. TheDNA molecules can escape this compartment with the help ofdioleoylethanolamine (DOPE) and are transported into the nucleus wherethey can be transcribed (Tranchant et al., 2004, Physicochemicaloptimisation of plasmid delivery by cationic lipids, J Gene Med 6 Suppl1:S24-35).

Immunoliposomes are liposomes with specific antibodies inserted intotheir membranes. The antibodies bind selectively to specific surfacemolecules on the target cell to facilitate uptake. The surface moleculestargeted by the antibodies are those that are preferably internalized bythe cells so that upon binding, the whole complex is taken up. Thisapproach increases the efficiency of transfection by enhancing theintracellular release of liposomal components. These antibodies can beinserted in the liposomal surface through various lipid anchors orattached at the terminus of polyethylene glycol grafted onto theliposomal surface. In addition to providing specificity to genedelivery, the antibodies can also provide a protective covering to theliposomes that helps to limit their degradation after uptake byendogenous RNAses or proteinases (Bendas, 2001, Immunoliposomes: Apromising approach to targeting cancer therapy, BioDrugs 15(4):215-224).To further prevent degradation of liposomes and their contents in thelysosomal compartment, pH sensitive immunoliposomes can be employed(Torchilin et al., 2006, pH-sensitive liposomes, J Liposome Res3:201-255). These liposomes enhance the release of liposomal contentinto the cytosol by fusing with the endosomal membrane within theorganelle as they become destabilized and prone to fusion at acidic pH.

In general, non-viral gene delivery systems have not been as widelyapplied as a means of gene delivery into stem cells as viral genedelivery systems. However, promising results are demonstrated in a studylooking at the transfection viability, proliferation and differentiationof adult neural stem/progenitor cells into the three neural lineagesneurons. Non-viral, non-liposomal gene delivery systems (ExGen500 andFuGene6) had a transfection efficiency of between 16% (ExGen500) and 11%(FuGene6) of cells. FuGene6-treated cells did not differ fromuntransfected cells in their viability or rate of proliferation, whereasthese characteristics were significantly reduced following ExGen500transfection. Importantly, neither agent affected the pattern ofdifferentiation following transfection. Both agents could be used togenetically label cells, and track their differentiation into the threeneural lineages, after grafting onto ex vivo organotypic hippocampalslice cultures (Tinsley et al, 2006, Efficient non-viral transfection ofadult neural stem/progenitor cells, without affecting viability,proliferation or differentiation, J Gene Med 8(1):72-81).

(iv) Polymer-Based Methods

The protonated epsilon-amino groups of poly L-lysine (PLL) interact withthe negatively charged DNA molecules to form complexes that can be usedfor gene delivery. These complexes can be rather unstable and showed atendency to aggregate. The conjugation of polyethylene glycol (PEG) wasfound to lead to an increased stability of the complexes. To confer adegree of tissue-specificity, targeting molecules such astissue-specific antibodies have also been employed. An additional genecarrier that has been used for transfecting cells is polyethylenimine(PEI) which also forms complexes with DNA. Due to the presence of amineswith different pKa values, it has the ability to escape the endosomalcompartment. PEG grafted onto PEI complexes was found to reduce thecytotoxicity and aggregation of these complexes. This can also be usedin combination with conjugated antibodies to confer tissue-specificity.See Lee & Kim, 2014, Bioreducible polymers for therapeutic genedelivery, J Control Relase ePub; Wang et al., 2013, Non-viral genedelivery methods, Curr Pharm Biotechnol 14(1):46-40; and Gupta et al.,2012, Structuring polymers for delivery of DNA-based therapeutics:updated insights, Crit Rev Ther Drug Carrier Syst 29(6):447-85.

Optical actuators, reporters, or both as discussed herein may betargeted to intracellular organelles, including mitochondria, theendoplasmic reticulum, the sarcoplasmic reticulum, synaptic vesicles,and phagosomes. Accordingly, in one embodiment, the invention providesexpression constructs, such as viral constructs comprising a reporterand/or actuatory operably linked to a sequence targeting the protein toan intracellular organelle, including a mitochondrion, an endoplasmicreticulum, a sarcoplasmic reticulum, a synaptic vesicle, and aphagosome. In some embodiments, the optical voltage sensor furthercomprises a localization or targeting sequence to direct or sort thesensor to a particular face of a biological membrane or subcellularorganelle. Preferred localization sequences provide for highly specificlocalization of the protein, with minimal accumulation in othersubcellular compartments. Localization signals are described in, e.g.,“Protein Targeting”, chapter of Stryer, L., Biochemistry (4th ed.). W.H.Freeman, 1995 and Chapter 12 (pages 551-598) of Molecular Biology of theCell, Alberts et al. third edition, (1994) Garland Publishing Inc. Insome embodiments, more than one discrete localization motif is used toprovide for correct sorting by the cellular machinery. For example,correct sorting of proteins to the extracellular face of the plasmamembrane can be achieved using an N-terminal signal sequence and aC-terminal GPI anchor or transmembrane domain.

Typically, localization sequences can be located almost anywhere in theamino acid sequence of the protein. In some cases the localizationsequence can be split into two blocks separated from each other by avariable number of amino acids. The creation of such constructs viastandard recombinant DNA approaches is well known in the art, as forexample described in Maniatis, et al., Molecular Cloning A LaboratoryManual, Cold Spring Harbor Laboratory, N. Y, 1989).

Methods of the invention can be used to express proteins transiently,stably, or both. Transduction and transformation methods for transientexpression of nucleic acids are well known to one skilled in the art.Transient transfection can be carried out, e.g., using calciumphosphate, by electroporation, or by mixing a cationic lipid with thematerial to produce liposomes, cationic polymers or highly branchedorganic compounds. All these are in routine use in genetic engineering.

Exemplary protocols for stable expression can be found, e.g., inEssential Stem Cell Methods, edited by Lanza and Klimanskaya, publishedin 2008, Academic Press. For example, one can generate a virus thatintegrates into the genome and comprises a selectable marker, and infectthe cells with the virus and screen for cells that express the marker,which cells are the ones that have incorporated the virus into theirgenome. A VSV-g psuedotyped lenti virus with a puromycin selectablemarker in HEK cells can be used according to established procedures.Generally, one can use a stem cell specific promoter to encode a GFP ifFACS sorting is necessary. The hiPS cultures are cultivated on embryonicfibroblast (EF) feeder layers or on Matrigel in fibroblast growth factorsupplemented EF conditioned medium. The cells are dissociated bytrypsinization, plated, and maintained in an undifferentiated state,e.g., using EF conditioned medium. Cells are cultured with the virus for24 hours; washed, typically with PBS, and fresh media is added with aselection marker, such as 1 micro g/mL puromycin. The medium is replacedabout every 2 days with additional puromycin. Cells surviving after 1week are re-plated, e.g., using the hanging drop method to form EBs withstable incorporation of gene.

In some embodiments, it is advantageous to express an optical voltagereporter (e.g., Arch D95N or a suitable variant thereof) in only asingle cell-type within an organism, and further, if desired, to directthe reporter to a particular subcellular structure within the cell.Upstream promoters control when and where the gene is expressed.Constructs are made that optimize expression in all eukaryotic cells. Inone embodiment, the optical voltage sensor is under the control of aneuron-specific promoter.

The promoter sequence can be selected to restrict expression of theprotein to a specific class of cells and environmental conditions.Common promoter sequences include, but are not limited to, CMV(cytomegalovirus promoter; a universal promoter for mammalian cells),14× UAS-E1b (in combination with the transactivator Gal4, this promoterallows combinatorial control of transgene expression in a wide array ofeukaryotes. Tissue-specific expression can be achieved by placing Gal4under an appropriate promoter, and then using Gal4 to drive theUAS-controlled transgene), HuC (drives pan-neuronal expression inzebrafish and other teleosts), ara (allows regulation of expression witharabinose in bacteria) and lac (allows regulation of expression withIPTG in bacteria).

Methods of the invention can be used to target actuators, reporters, orboth to specific cellular sites such as the plasma membrane. In someembodiments, constructs are designed to include signaling sequences tooptimize localization of the protein to the plasma membrane. These caninclude e.g., a C-terminal signaling sequence from the O.sub.2 nicotinicacetylcholine receptor and/or an endoplasmic reticulum export motif fromKir2.1.

Additional improvements in plasma localization can be obtained by addingGolgi export sequences and membrane localization sequences. See Gong etal., 2014, Imaging neural spiking in brain tissue using FRET-opsinprotein voltage sensors, Nat Comm 5:articel3674; and Gradinaru et al.,2010, Molecular and Cellular Approaches for Diversifying and ExtendingOptogenetics, Cell 141:154-165. In some embodiments, the targetingsequence is selected to regulate intracellular transport of the proteinto the desired subcellular structure. In one embodiment the protein istargeted to the plasma membrane of a eukaryotic cell. In this case thetargeting sequence can be designed following the strategy outlined ine.g., Gradinaru 2010. The term “signal sequence” refers to N-terminaldomains that target proteins into a subcellular locale e.g., theendoplasmic reticulum (ER), and thus are on their way to the plasmamembrane. Signal sequences used in optogenetic voltage sensors can bederived from the proteins beta-2-n-acetylcholine receptor (SS B2nAChR)and PPL. In addition, there is an endogenous signaling sequence onmicrobial rhodopsin proteins that can be harnessed for appropriatesubcellular targeting. A trafficking signal (TS) can optionally beinserted into the genome C-terminal to the microbial rhodopsin andN-terminal to the accessory fluorescent protein. In one embodiment, thetrafficking signal is derived from the Kir2.1 protein as specified inGradinaru et al. In another embodiment, an ER export motif is insertedat the C-terminus of the accessory fluorescent protein.

A construct of the invention may be localized to the mitochondrial innermembrane or mitochondrial outer membrane, i.e., using appropriatesignaling sequences added to the rhodopsin protein. Optogeneticreporters can be targeted to the inner mitochondrial membrane as thatdescribed in Hoffmann et al., 1994, Photoactive mitochondria: in vivotransfer of a light-driven proton pump into the inner mitochondrialmembrane of Schizosaccharomyces pombe, PNAS 91:9367-9371.

As discussed above, the invention includes optogenetic reporters,optogenetic actuators, and vectors for the expression of microbialrhodopsins. See also U.S. Pat. No. 8,716,447 to Deisseroth; U.S. Pat.No. 8,647,870 to Hegemann; U.S. Pat. No. 8,617,876 to Farrar; U.S. Pat.No. 8,603,790 to Deisseroth; U.S. Pat. No. 8,580,937 to Spudich; U.S.Pat. No. 8,562,658 to Shoham; and U.S. Pat. No. 8,202,699 to Hegemann,the contents of each of which are incorporated by reference.

The invention further provides cells expressing the constructs, andfurther methods of measuring membrane potential changes in the cellsexpressing such constructs as well as methods of screening for agentsthat affect the membrane potential of one or more of the intracellularmembranes.

5. Imaging Activity Assay

5a. Capturing Images

Methods of the invention may include stimulating the cells that are tobe observed. Stimulation may be direct or indirect (e.g., opticalstimulation of an optical actuator or stimulating an upstream cell insynaptic or gap junction-mediated communication with the cell(s) to beobserved). Stimulation may be optical, electrical, chemical, or by anyother suitable method. Stimulation may involve any pattern of astimulation including, for example, regular, periodic pulses, singlepulses, irregular patterns, or any suitable pattern. Methods may includevarying optical stimulation patterns in space or time to highlightparticular aspects of cellular function. For example, a pulse patternmay have an increasing frequency. In certain embodiments, imagingincludes stimulating a neuron that expresses an optical actuator usingpulses of light.

Optical reporters of the invention provide accurate values of themembrane potential, without systematic artifacts from photobleaching,variation in illumination intensity, cell movement, or variations inprotein expression level. In cells that are accessible to patch clamp,one can calibrate the fluorescence as a function of membrane potentialby varying the membrane potential under external control. However,constructs of the invention function in systems that are inaccessible topatch clamp. In these cases direct calibration is not possible.

The Arch 3 fusion with eGFP enables ratiometric determination ofmembrane potential. Similar ratiometric determinations may be made usingother optical reporters such as those described in this applicationusing the identical concept. The eGFP fluorescence is independent ofmembrane potential, The ratio of Arch 3 fluorescence to eGFPfluorescence provides a measure of membrane potential that isindependent of variations in expression level, illumination, ormovement.

In the methods of the invention, the cells are excited with a lightsource so that the emitted fluorescence can be detected. The wavelengthof the excitation light depends on the fluorescent molecule. Forexample, the Archaerhodopsin constructs in the examples are allexcitable using light with wavelengths varying between lambda=594 nm andlambda=645 nm. Alternatively, the range may be between lambda=630-645nm. For example a commonly used Helium Neon laser emits at lambda=632.8nm and can be used in excitation of the fluorescent emission of thesemolecules.

In some embodiments a second light is used. For example, if the cellexpresses a reference fluorescent molecule or a fluorescent moleculethat is used to detect another feature of the cell, such a pH or Calciumconcentration. In such case, the second wavelength differs from thefirst wavelength. Examples of useful wavelengths include wavelengths inthe range of lambda=447-594 nm, for example, lambda=473 nm, lambda=488nm, lambda=514 nm, lambda=532 nm, and lambda=561 nm.

Methods of the invention allow for the measurement of action potentialswith sub-millisecond temporal resolution. A neuron expressing anOptopatch construct may be exposed to whole-field illumination withpulses of blue light (10 ms, 25 mW/cm²) to stimulate CheRiff, andsimultaneous constant illumination with red light (800 W/cm²) to excitefluorescence of the reporter (e.g., Arch D95N or a suitable variantthereof). The fluorescence of the reporter may be imaged at a 1 kHzframe rate. Key parameters include temporal precision with which singlespikes can be elicited and recorded, signal-to-noise ratio (SNR) influorescence traces, and long-term stability of the reporter signal.Methods provided herein may be found to optimize those parameters.Further discussion may be found in Foust et al., 2010, Action potentialsinitiate in the axon initial segment and propagate through axoncollaterals reliably in cerebellar Purkinje neurons, J. Neurosci30:6891-6902; and Popovic et al., 2011, The spatio-temporalcharacteristics of action potential initiation in layer 5 pyramidalneurons: a voltage imaging study, J. Physiol. 589:4167-4187.

In some embodiments, measurements are made using a low-magnificationmicroscope that images a 1.2×3.3 mm field of view with 3.25 μm spatialresolution and 2 ms temporal resolution. In other embodiments,measurements are made using a high-magnification microscope that imagesa 100 μm field of view with 0.8 μm spatial resolution and 1 ms temporalresolution. A suitable instrument is an inverted fluorescencemicroscope, similar to the one described in the Supplementary Materialto Kralj et al., 2012, Optical recording of action potentials inmammalian neurons using a microbial rhodopsin, Nat. Methods 9:90-95.Briefly, illumination from a red laser 640 nm, 140 mW (Coherent Obis637-140 LX), is expanded and focused onto the back-focal plane of a 60×oil immersion objective, numerical aperture 1.45 (Olympus 1-U2B616).

FIG. 10 gives a functional diagram of components of an optical imagingapparatus 501 according to certain embodiments. A 488 nm blue laser beamis modulated in intensity by an acousto-optic modulator (not shown), andthen reflected off a digital micromirror device (DMD) 505. The DMDimparted a spatial pattern on the blue laser beam (used for CheRiffexcitation) on its way into the microscope. The micromirrors werere-imaged onto the sample 509, leading to an arbitrary user-definedspatiotemporal pattern of illumination at the sample. Simultaneouswhole-field illumination with 640 nm red light excites fluorescence ofthe reporter.

With the inverted fluorescence microscope, illumination from a bluelaser 488 nm 50 mW (Omicron PhoxX) is sent through an acousto-opticmodulator (AOM; Gooch and Housego 48058-2.5-.55-5W) for rapid controlover the blue intensity. The beam is then expanded and modulated by DMD505 with 608×684 pixels (Texas Instruments LightCrafter). The DMD iscontrolled via custom software (Matlab) through a TCP/IP protocol. TheDMD chip is re-imaged through the objective onto the sample, with theblue and red beams merging via a dichroic mirror. Each pixel of the DMDcorresponds to 0.65 μm in the sample plane. A 532 nm laser is combinedwith the red and blue beams for imaging of mOrange2. Software is writtento map DMD coordinates to camera coordinates, enabling precise opticaltargeting of any point in the sample.

To achieve precise optical stimulation of user-defined regions of aneuron, pixels on DMD 505 are mapped to pixels on the camera. The DMDprojects an array of dots of known dimensions onto the sample. Thecamera acquires an image of the fluorescence. Custom software locatesthe centers of the dots in the image, and creates an affinetransformation to map DMD coordinates onto camera pixel coordinates.

A dual-band dichroic filter (Chroma zt532/635rpc) separates reporter(e.g., Arch) from excitation light. A 531/40 nm bandpass filter (SemrockFF01-531/40-25) may be used for eGFP imaging; a 710/100 nm bandpassfilter (Chroma, HHQ710/100) for Arch imaging; and a quad-band emissionfilter (Chroma ZET405/488/532/642m) for mOrange2 imaging andpre-measurement calibrations. A variable-zoom camera lens (Sigma 18-200mm f/3.5-6.3 II DC) is used to image the sample onto an EMCCD camera(Andor iXon+DU-860), with 128×128 pixels. Images may be first acquiredat full resolution (128×128 pixels). Data is then acquired with 2×2pixel binning to achieve a frame rate of 1,000 frames/s. For runs withinfrequent stimulation (once every 5 s), the red illumination is only onfrom 1 s before stimulation to 50 ms after stimulation to minimizephotobleaching. Cumulative red light exposure may be limited to <5 min.per neuron.

Low magnification wide-field imaging is performed with a custommicroscope system based around a 2×, NA 0.5 objective (Olympus MVX-2).Illumination is provided by six lasers 640 nm, 500 mW (Dragon Lasers635M500), combined in three groups of two. Illumination is coupled intothe sample using a custom fused silica prism, without passing throughthe objective. Fluorescence is collected by the objective, passedthrough an emission filter, and imaged onto a scientific CMOS camera(Hamamatsu Orca Flash 4.0). Blue illumination for channelrhodopsinstimulation is provided by a 473 nm, 1 W laser (Dragon Lasers),modulated in intensity by an AOM and spatially by a DMD (Digital LightInnovations DLi4130—ALP HS). The DMD is re-imaged onto the sample viathe 2× objective. During a run, neurons may be imaged using wide-fieldillumination at 488 nm and eGFP fluorescence. A user may select regionsof interest on the image of the neuron, and specify a time course forthe illumination in each region. The software maps the user-selectedpixels onto DMD coordinates and delivers the illumination instructionsto the DMD.

The inverted fluorescence micro-imaging system records optically fromnumerous (e.g., 50) expressing cells or cell clusters in a single fieldof view. The system may be used to characterize optically evoked firingpatterns and AP waveforms in neurons expressing an Optopatch construct.Each field of view is exposed to whole-field pulses of blue light toevoke activity (e.g., 0.5 s, repeated every 6 s, nine intensitiesincreasing from 0 to 10 mW/cm²). Reporter fluorescence such as from ArchD95N may be simultaneously monitored with whole-field excitation at 640nm, 100 W/cm². Additional useful discussion of microscopes and imagingsystems may be found in U.S. Pat. No. 8,532,398 to Filkins; U.S. Pat.No. 7,964,853 to Araya; U.S. Pat. No. 7,560,709 to Kimura; U.S. Pat. No.7,459,333 to Richards; U.S. Pat. No. 6,972,892 to DeSimone; U.S. Pat.No. 6,898,004 to Shimizu; U.S. Pat. No. 6,885,492 to DeSimone; and U.S.Pat. No. 6,243,197 to Schalz, the contents of each of which areincorporated by reference.

FIG. 11 illustrates a pulse sequence of red and blue light used torecord action potentials under increasing optical stimulation. In someembodiments, neurons are imaged on a high resolution microscope with 640nm laser (600 W/cm²) for voltage imaging. In certain embodiments,neurons are imaged on a high resolution microscope with 640 nm laser(600 W/cm²) for voltage imaging and excited with a 488 nm laser (20-200mW/cm²). Distinct firing patterns can be observed (e.g., fast adaptingand slow-adapting spike trains). System measurements can detect rareelectrophysiological phenotypes that might be missed in a manual patchclamp measurement. Specifically, the cells' response to stimulation(e.g., optical actuation) may be observed. Instruments suitable for useor modification for use with methods of the invention are discussed inU.S. Pub. 2013/0170026 to Cohen, incorporated by reference.

Using the described methods, populations of cells may be measured. Forexample, both diseased and corrected (e.g., by zing finger domains)motor neurons may be measured. A cell's characteristic signature such asa neural response as revealed by a spike train may be observed.

5b. Extracting Fluorescence from Movies

Fluorescence values are extracted from raw movies by any suitablemethod. One method uses the maximum likelihood pixel weighting algorithmdescribed in Kralj et al., 2012, Optical recording of action potentialsin mammalian neurons using a microbial rhodopsin, Nat Methods 9:90-95.Briefly, the fluorescence at each pixel is correlated with thewhole-field average fluorescence. Pixels that showed strongercorrelation to the mean are preferentially weighted. This algorithmautomatically finds the pixels carrying the most information, andde-emphasizes background pixels.

In movies containing multiple cells, fluorescence from each cell isextracted via methods known in the art such as Mukamel et al., 2009,Automated analysis of cellular signals from large-scale calcium imagingdata, Neuron 63(6):747-760, or Maruyama et al., 2014, Detecting cellsusing non-negative matrix factorization on calcium imaging data, NeuralNetworks 55:11-19. These methods use the spatial and temporalcorrelation properties of action potential firing events to identifyclusters of pixels whose intensities co-vary, and associate suchclusters with individual cells.

Alternatively, a user defines a region comprising the cell body andadjacent neurites, and calculates fluorescence from the unweighted meanof pixel values within this region. In low-magnification images, directaveraging and the maximum likelihood pixel weighting approaches may befound to provide optimum signal-to-noise ratios.

6. Signal Processing

6a. Signal Processing with Independent Component Analysis to AssociateSignals with Cells

An image or movie may contain multiple cells in any given field of view,frame, or image. In images containing multiple neurons, the segmentationis performed semi-automatically using an independent components analysis(ICA) based approach modified from that of Mukamel, et al., 2009,Automated analysis of cellular signals from large-scale calcium imagingdata, Neuron 63:747-760. The ICA analysis can isolate the image signalof an individual cell from within an image.

FIG. 12-FIG. 15 illustrate the isolation of individual cells in a fieldof view. Individual cells are isolated in a field of view using anindependent component analysis.

FIG. 12 shows an image that contains five neurons whose images overlapwith each other. The fluorescence signal at each pixel is an admixtureof the signals from each of the neurons underlying that pixel.

As shown in FIG. 13, the statistical technique of independent componentsanalysis finds clusters of pixels whose intensity is correlated within acluster, and maximally statistically independent between clusters. Theseclusters correspond to images of individual cells comprising theaggregate image of FIG. 12.

From the pseudo-inverse of the set of images shown in FIG. 13 arecalculated spatial filters with which to extract the fluorescenceintensity time-traces for each cell. Filters are created by setting allpixel weights to zero, except for those in one of the image segments.These pixels are assigned the same weight they had in the original ICAspatial filter.

In FIG. 14, by applying the segmented spatial filters to the movie data,the ICA time course has been broken into distinct contributions fromeach cell. Segmentation may reveal that the activities of the cells arestrongly correlated, as expected for cells found together by ICA. Inthis case, the spike trains from the image segments are similar but showa progress corresponding to different physiological responses of thecells to the stimulus pattern shown in FIG. 11.

FIG. 15 shows an overlay of the individual filters used to map (andcolor code) individual cells from the original image.

6b. Signal Processing Via Sub-Nyquist Action Potential Timing (SNAPT)

For individual cells, the sub-cellular details of action potentialpropagation can be represented by the timing at which an interpolatedaction potential crosses a threshold at each pixel in the image.Identifying the wavefront propagation may be aided by first processingthe data to remove noise, normalize signals, improve SNR, otherpre-processing steps, or combinations thereof. Action potential signalsmay first be processed by removing photobleaching, subtracting a medianfiltered trace, and isolating data above a noise threshold. The APwavefront may then be identified using an algorithm based on sub-Nyquistaction potential timing such as an algorithm based on the interpolationapproach of Foust, et al., 2010, Action potentials initiate in the axoninitial segment and propagate through axon collaterals reliably incerebellar Purkinje neurons. J. Neurosci 30, 6891-6902 and Popovic etal, 2011, The spatio-temporal characteristics of action potentialinitiation in layer 5 pyramidal neurons: a voltage imaging study. J.Physiol. 589, 4167-4187.

A sub-Nyquist action potential timing (SNAPT) algorithm highlightssubcellular timing differences in AP initiation. For example, thealgorithm may be applied for neurons expressing Optopatch1, containing avoltage reporter such as Arch D95N or a suitable variant thereof and avoltage actuator such as CheRiff. Either the soma or a small dendriticregion is stimulated via repeated pulses of blue light. The timing andlocation of the ensuing APs is monitored.

FIG. 16 shows a patterned optical excitation being used to induce actionpotentials. Movies of individual action potentials are acquired (e.g.,at 1,000 frames/s), temporally registered, and averaged.

The first step in the temporal registration of spike movies is todetermine the spike times. Determination of spike times is performediteratively. A simple threshold-and-maximum procedure is applied to thewhole-field fluorescence trace, F(t), to determine approximate spiketimes, {T0}. Waveforms in a brief window bracketing each spike areaveraged together to produce a preliminary spike kernel K₀(t). Across-correlation of K₀(t) with the original intensity trace F(t) iscalculated. Whereas the timing of maxima in F(t) is subject to errorsfrom single-frame noise, the peaks in the cross-correlation, located attimes {T}, are a robust measure of spike timing. A movie showing themean AP propagation may be constructed by averaging movies in briefwindows bracketing spike times {T}. Typically 100-300 APs are includedin this average. The AP movie has high signal-to-noise ratio. Areference movie of an action potential is thus created by averaging thetemporally registered movies (e.g., hundreds of movies) of single APs.

Spatial and temporal linear filters may further decrease the noise in APmovie. A spatial filter may include convolution with a Gaussian kernel,typically with a standard deviation of 1 pixel. A temporal filter may bebased upon Principal Components Analysis (PCA) of the set ofsingle-pixel time traces. The time trace at each pixel is expressed inthe basis of PCA eigenvectors. Typically the first 5 eigenvectors aresufficient to account for >99% of the pixel-to-pixel variability in APwaveforms, and thus the PCA eigen-decomposition is truncated after 5terms. The remaining eigenvectors represented uncorrelated shot noise.

FIG. 17 shows eigenvectors resulting from a principal component analysis(PCA) smoothing operation performed to address noise. Photobleaching orother such non-specific background fluorescence may be addressed bythese means.

FIG. 18 shows a relation between cumulative variance and eigenvectornumber. FIG. 19 gives a comparison of action potential waveforms beforeand after the spatial and PCA smoothing operations.

A smoothly varying spline function may be interpolated between thediscretely sampled fluorescence measurements at each pixel in thissmoothed reference AP movie. The timing at each pixel with which theinterpolated AP crosses a user-selected threshold may be inferred withsub-exposure precision. The user sets a threshold depolarization totrack (represented as a fraction of the maximum fluorescence transient),and a sign for dV/dt (indicating rising or falling edge. The filtereddata is fit with a quadratic spline interpolation and the time ofthreshold crossing is calculated for each pixel.

FIG. 20 shows an action potential timing map. The timing map may beconverted into a high temporal resolution SNAPT movie by highlightingeach pixel in a Gaussian time course centered on the local AP timing.The SNAPT fits are converted into movies showing AP propagation asfollows. Each pixel is kept dark except for a brief flash timed tocoincide with the timing of the user-selected AP feature at that pixel.The flash followed a Gaussian time-course, with amplitude equal to thelocal AP amplitude, and duration equal to the cell-average timeresolution, σ. Frame times in the SNAPT movies are selected to be˜2-fold shorter than a. Converting the timing map into a SNAPT movie isfor visualization; propagation information is in the timing map.

FIG. 21 shows the accuracy of timing extracted by the SNAPT algorithmfor voltage at a soma via comparison to a simultaneous patch clamprecording. FIG. 22 gives an image of eGFP fluorescence, indicatingCheRiff distribution.

FIG. 23 presents frames from a SNAPT movie formed by mapping the timinginformation from FIG. 20 onto a high spatial resolution image from FIG.22. In FIG. 23, the white arrows mark the zone of action potentialinitiation in the presumed axon initial segment (AIS). FIGS. 20-23demonstrate that methods of the invention can provide high resolutionspatial and temporal signatures of cells expressing an optical reporterof neural activity.

After acquiring Optopatch data, cells may be fixed and stained forankyrin-G, a marker of the axon initial segment (AIS). Correlation ofthe SNAPT movies with the immunostaining images establish that the APinitiated at the distal end of the AIS. The SNAPT technique does notrely on an assumed AP waveform; it is compatible with APs that changeshape within or between cells.

The SNAPT movies show AP initiation from the soma in single neurites inmeasured cells. The described methods are useful to reveal latenciesbetween AP initiation at the AIS and arrival in the soma of 320±220 μs,where AP timing is measured at 50% maximum depolarization on the risingedge. Thus Optopatch can resolve functionally significant subcellulardetails of AP propagation. Discussion of signal processing may be foundin Mukamel et al., 2009, Automated analysis of cellular signals fromlarge-scale calcium imaging data, Neuron 63(6):747-760.

Methods of the invention are used to obtain a signature from theobserved cell or cells tending to characterize a physiological parameterof the cell. The measured signature can include any suitableelectrophysiology parameter such as, for example, activity at baseline,activity under different stimulus strengths, tonic vs. phasic firingpatterns, changes in AP waveform, others, or a combination thereof.Measurements can include different modalities, stimulation protocols, oranalysis protocols. Exemplarily modalities for measurement includevoltage, calcium, ATP, or combinations thereof. Exemplary stimulationprotocols can be employed to measure excitability, to measure synaptictransmission, to test the response to modulatory chemicals, others, andcombinations thereof. Methods of invention may employ various analysisprotocols to measure: spike frequency under different stimulus types,action potential waveform, spiking patterns, resting potential, spikepeak amplitude, others, or combinations thereof.

In certain embodiments, the imaging methods are applied to obtain asignature mean probability of spike for cells from a subject and mayalso be used to obtain a signature from a control line of cells such asa wild-type control (which may be produced by genome editing asdescribed above so that the control and the wild-type are isogenic butfor a single site). The observed signature can be compared to a controlsignature and a difference between the observed signature and theexpected signature corresponds to a characteristic of the cell.

FIG. 24 shows a mean probability of spike of wild-type (WT) and mutant(SOD1 A4V) motor neurons derived from human induced pluripotent stemcells. The SOD1 A4V mutations is associated with amyotrophic lateralsclerosis (ALS). Cellular excitability was measured by probability ofspiking during each blue light stimulation, and during no stimulation(spontaneous firing). The mutant neurons had increased rate of firing inthe absence of optical stimulation, but a decreased rate of firing understrong optical stimulation.

8. Additional Methods & Applications

Methods of the invention may include the use of tool/test compounds orother interventional tools applied to the observed cell or cells.Application of test compounds can reveal effects of those compounds oncellular electrophysiology. Use of tool compounds can achieve greaterspecificity in diagnosis or for determining disease mechanisms, e.g. byblocking certain ion channels. By quantifying the impact of thecompound, one can quantify the level of that channel in the cell.

With a tool or test compound, a cell may be caused to express an opticalreporter of neural or electrical activity and may also be exposed to acompound such as a drug. A signature of the cell can be observed before,during, or after testing the compound. Any combination of differentcells and cell types can be exposed to one or any combination ofcompounds, including different test compound controls. Multi-wellplates, multi-locus spotting on slides, or other multi-compartment labtools can be used to cross-test any combination of compounds and celltypes.

In certain embodiments, tool compounds are added to cells and theireffect on the cells is observed to distinguish possible diseases orcauses or mechanisms of diseases. For example, where two or more cellsin synaptic connection with one another are observed, extrinsicstimulation of an upstream cell should manifest as an action potentialin a downstream cell. A compound that is known to inhibitneurotransmitter reuptake may be revealed to work on only certain neuralsubtypes thus indicating a specific disease pattern.

In some embodiments, methods of the invention are used to detect,measure, or evaluate synaptic transmission. A signature may be observedfor a cell other than the cell to which direct stimulation was applied.In fact, using the signal processing algorithms discussed herein,synaptic transmission among a plurality of cells may be detected thusrevealing patterns of neural connection. Establishing an assay thatsuccessfully detects firing of a downstream neuron upon stimulation ofan upstream neuron can reveal, where the subject cell to be observedfails to fire upon stimulation of an upstream neuron, a disease orcondition characterized by a failure of synaptic transmission.

Test compounds can be evaluated as candidate therapies to determinesuitability of a treatment prior to application to a patient. E.g. onecan test epilepsy drugs to find the one that reverts the firing patternback to wild-type. In some embodiments, the invention provides systemsand methods for identifying possible therapies for a patient by testingcompounds, which systems and methods may be employed as personalizedmedicine. Due to the nature of the assays described herein, it may bepossible to evaluate the effects of candidate therapeutic compounds on aper-patient basis thus providing a tool for truly personalized medicine.For example, an assay as described herein may reveal that a patientsuffering from a certain disease has neurons or neural subtypes thatexhibit a disease-type physiological phenotype under the assaysdescribed herein. One or a number of different compounds may be appliedto those neurons or neural subtypes. Cells that are exposed to one ofthose different compounds (or a combination of compounds) may exhibit achange in physiological phenotype from disease-type to normal. Thecompound or combination of compounds that affects the change inphenotype from disease-type to normal is thus identified as a candidatetreatment compound for that patient.

Provided herein are areas in which an improved optical voltage indicatorcan be applied both in commercial and scientific endeavors.

Network Effects

Systems and methods of the invention may be used to study and usenetwork effects, e.g. where one set or class of neurons orcardiomyocytes contains the actuator and a different set or class ofcells (either intermixed or adjacent) contains the reporter. Morespecifically, both cell sets might have both actuator and reporter; orone set might have the actuator only, and the other set may have thereporter only. This ability to probe network effects may be particularlyimportant as many genes, such as ones that are being implicated inschizophrenia and bipolar disorder, code for synaptic proteins. Seebackground discussion neural activity in U.S. Pat. No. 8,401,609 toDeisseroth, the contents of which are incorporated by reference.

Network effects also promise to be important in the cardiac area, wherefor example a monolayer of cardiomyocytes may be illuminated with somecells expressing actuators of the invention while imaged via othersexpressing the reporters. Cells of the invention (e.g., neurons,cardiomyocytes, etc.) may be visualized via a microscope of theinvention. Those cells may be in electrical or synaptic communicationwith one another.

Additionally, it is noted that where networks of cells signal, thesignals may propagate from cell to cell in a one-to-one, one-to-many,many-to-one, many-to-many schema, or a combination thereof. That is,axon terminals of two or more neurons may be in synaptic communicationwith dendrites of one or more other neurons. Where a plurality of cellsform a network, signal processing described in section 6 above may beemployed to discern which individual cells have signaled which, when,and how quickly. Thus systems and methods of the invention may be usedto—for example—study, discover, or diagnose a condition affecting asynaptic protein.

In some embodiments, the invention provides a method in which one firstset of cells each includes an actuator and a second set of cells eachincludes an optical reporter. The method includes stimulating the firstset of cells and measuring a signal from the optical reporter, therebyevaluating whether cells of the first set of cells transmitted a signalto cells of the second set of cells. Preferably, the actuator is anoptical actuator such as CheRiff and stimulating the first set of cellsincludes illuminating the CheRiff actuator.

Cardiomyocytes

Methods and systems of the invention may be used to characterize cardiaccells. A cell can be obtained and converted into a cardiomyocyte. Forexample, using methods described herein, fibroblasts may be converted tocardiomyocytes via induced pluripotent stem cells. An optical actuatorof electrical activity, an optical reporter of electrical activity, orboth may be incorporated into any one or more of cardiomyocytes asdescribed above. As shown in FIGS. 24-30, a signal may be obtained fromthe optical reporter in response to a stimulation of the cardiomyocytes.By evaluating the signal, the cardiomyocytes are characterized.

FIG. 25 demonstrates effects of DMSO vehicle control on the actionpotential (AP) waveforms of hiPSC-derived cardiomyocytes. Representativesegments of the mean fluorescence (ΔF/F) versus time (seconds, s) tracesat each concentration (0% ‘blank’, 0.003%, 0.01%, 0.03%, 0.1% and 0.3%DMSO) are shown for spontaneously beating cells (top panel) as well asthe same cells optogenetically paced at 1 Hz (middle panel) and 2 Hz(bottom panel). Traces are taken from a single dish of cells and asingle field-of-view. Data was taken at 100 Hz frame rate.

FIG. 26 presents the effects of DMSO control vehicle on the average APwaveform. The average waveform for the range of concentrations tested(cyan to magenta; lowest to highest concentrations tested, respectively)is shown. The top, middle, and bottom panels correspond to spontaneousbeating, 1 Hz pacing and 2 Hz pacing, respectively. Dashed linesindicate that the cells did not beat at the specified pacing rate. Inthe case of spontaneous beating, this criterion did not apply. Panelsare calculated from data taken at 100 Hz.

FIG. 27 presents the effects of DMSO control vehicle on the average risetime. The average rise time for the range of concentrations tested (cyanto magenta; lowest to highest concentrations tested, respectively) isshown. The top, middle, and bottom panels correspond to spontaneousbeating, 1 Hz pacing and 2 Hz pacing, respectively. Dashed linesindicate that the cells did not beat at the specified pacing rate. Inthe case of spontaneous beating, this criterion did not apply. Panelsare calculated from data taken at 500 Hz.

FIGS. 28-31 illustrate the quantification of the effect of DMSO additionon AP waveform.

FIG. 28 shows the dose dependence of the action potential duration at50% of repolarization (AP50).

FIG. 29 shows the dose dependence of the action potential duration at90% of repolarization (AP90).

FIG. 30 shows the dose dependence of the AP rise time.

FIG. 31 shows the dose dependence of the spontaneous beat rate.

In FIGS. 28-31, closed circles are used to represent the ‘blank’addition of imaging buffer alone whereas open circles are used torepresent the addition of compound at varying concentrations. Analysiswas performed on fluorescence versus time traces acquired underconditions of spontaneous beating (black) as well as pacing regimens of1 Hz (blue) and 2 Hz (red). Note that in the case of 1 Hz and 2 Hzpacing, data points are omitted from the plot in the event that thecells do not pace at the specified pace rate. Data points are alsoomitted in the event that the cells stop beating. Data and error barsare reported as the mean+/−standard error of the mean.

Brain Imaging

The human brain functions by sending electrical impulses along itsneurons. These patterns of firing are the origin of human thought andaction. The invention potentially provides methods for observinglarge-scale patterns of electrical activity in an intact brain. (Forsome background, see Baker et al., 2008, Genetically encoded fluorescentsensors of membrane potential, Brain Cell Biol 36(1-4):53-67.) Use ofoptical actuators and reporters of the invention may provideunprecedented insights in neuroscience. Methods and device of theinvention may allow mapping of brain activity in patients or cells ofpatients with psychiatric and neurological diseases, and in victims oftraumatic injuries or animal models modeling such diseases and injuries.

Optical imaging of neuronal activity can also form the basis forimproved brain-machine interfaces for people with disabilities. Forimaging in the brain, the optical reporter is administered by directinjection into the site to be analyzed (with or without accompanyingelectroporation) or the optical reporter is delivered using a viralvector. Alternatively the optical reporter may be administered throughthe formation of a transgenic organism, or through application of theCre-Lox recombination system.

Diagnosis

Methods of the invention may be used in the diagnosis of medicalconditions.

FIG. 31 illustrates an output from measuring action potentials in cellsaffected by a mutation and control cells isogenic but for the mutation.The recordings were acquired on motor neurons formed from hiPSCs andexpressing the CheRiff voltage actuator and an Arch-based voltagereporter. In the illustrated example, a patient known to have SOD1A4V—arisk factor for amyotrophic lateral sclerosis (ALS)—is studied and thebottom trace is obtained from cells of that patient's genotype. The toptrace labeled “WT” refers to cells from that patient that were edited tobe SOD1V4A and thus wild-type at the locus of the patient's knownmutation but otherwise to provide the genetic context present in thepatient. A clinician may diagnosis a neurodegenerative disease based ona signature spike train manifest by the patient's cells. Here, adifference between the signature observed in the patient's cells and thecontrol signature may be correlated to a positive diagnosis of aneurodegenerative disease.

Any suitable method of correlating the patient's signature to adiagnosis may be used. For example, in some embodiments, visualinspection of a signature may be used. In certain embodiments, acomputer system may be used to automatically evaluate that an observedsignature of the test cells satisfies predetermined criteria for adiagnosis. Any suitable criteria can be used. For example, a computersystem may integrate under the spike train for both the test cells andthe control cells over a range of time of at least a few seconds andcompare a difference between the results. Any suitable differencebetween the observed and expected signals can be used, for example, thedifference may include a modified probability of a voltage spike inresponse to the stimulation of the cell relative to a control. Incertain embodiments (e.g., FIG. 31) the difference between the observedsignal and the expected signal comprises a decreased probability of avoltage spike in response to the stimulation of the cell relative to acontrol and an increased probability of a voltage spike during periodsof no stimulation of the cell relative to a control. In one embodiment,systems and methods of the invention detect a decreased probability of avoltage spike in response to the stimulation of the cell relative to acontrol.

To give one example, a difference of at least 5% can be reported asindicative of an increased risk or diagnosis of a condition. In anotherexample, a computer system can analyze a probability of spike at acertain time point (e.g., 5500 ms) and look for a statisticallysignificant difference. In another example, a computer system can beprogrammed to first identify a maximal point in the WT spike train(control signature) and then compare a probability at that point in thecontrol signature to a probability in the patient's test signature atthe same point and look for a reportable difference (e.g., at least 5%different). One of skill in the art will recognize that any suitablecriterion can be used in the comparison of the test signature to thecontrol signature. In certain embodiments, a computer system is trainedby machine learning (e.g., numerous instances of known healthy and knowndiseased are input and a computer system measures an average differencebetween those or an average signature pattern of a disease signature).Where the computer system stores a signature pattern for a diseasephenotype, a diagnosis is supported when the computer system finds amatch between the test signature and the control signature (e.g., <5%different or less than 1% different at some point or as integrated overa distance). While obtaining a control signature from a genome-editedcell line from the patient has been discussed, one of skill in the artwill recognize that the control signature can be a template ordocumented control signature stored in computer system of the invention.

In certain embodiments, observation of a signature from a cell is usedin a diagnosis strategy in which the observed signature phenotypecontributes to arriving at a final diagnosis. For example, with certaindisease of the nervous system such as ALS, different neuron types may beaffected differently. In some embodiments, a diagnostic method includescomparing different neuron types from the same patient to diagnose asub-type specific disease.

Microbiology

Methods of the invention may be used in microbiology, for example, tostudy electrophysiology of microorganisms. Bacteria are host to dozensof ion channels of unknown function (Martinac et al., 2009, Ion channelsin microbes, Physiol Rev 88(4):1449). Most bacteria are too small fordirect electrophysiological measurements, so their electrical propertiesare almost entirely unknown. Upon expressing PROPS in E. coli, it wasfound that E. coli undergo a previously unknown electrical spikingbehavior. The data described herein in the Examples section is the firstreport of spontaneous electrical spiking in any bacterium. This resultestablishes the usefulness of voltage sensors in microbes.

Furthermore, studies with PROPS revealed that electrical spiking in E.coli is coupled to efflux of a cationic membrane permeable dye. It isthus plausible that electrical spiking is correlated to efflux of othercationic compounds, including antibiotics. Optical voltage indicatorsmay prove useful in screens for inhibitors of antibiotic efflux.

Optical voltage sensors will unlock the electrophysiology of themillions of species of microorganisms which have proven too small toprobe via conventional electrophysiology. This information will beuseful for understanding the physiology of bacteria with medical,industrial, and ecological applications. Additional discussion may befound in Kralj et al, 2011, Electrical spiking in Escherichia coliprobed with a fluorescent voltage-indicating protein, Science333(6040):345-348.

Mitochondria and Metabolic Diseases

Mitochondria are membrane-bound organelles which act as the ATPfactories in eukaryotic cells. A membrane voltage powers themitochondrial ATP synthase. Dysfunction of mitochondria has beenimplicated in a variety of neurodegenerative diseases, diabetes, cancer,cardiovascular disease, and aging. Thus there is tremendous interest inmeasuring mitochondrial membrane potential in vivo, although currentlyavailable techniques are severely limited (Verburg & Hollenbeck, 2009,Mitochondrial membrane potential in axons increases with local NGF orsemaphoring signaling, Neurosci. 28(33):8306-8315; Ichas et al., 1997,Mitochondria are excitable organelles capable of generating andconveying electrical and calcium signals, Cell 89(7):1145-1154.)

The exemplary optical voltage sensor described herein (PROPS) can betagged with peptide sequences that direct it to the mitochondrial innermembrane (Hoffmann et al., 1994, Photoactive mitochondria: In vivotransfer of a light-driven proton pump into the inner mitochondrialmembrane of Schizosaccharoyces pombe, PNAS 91:9367-9371) or themitochondrial outer membrane, where it serves as an optical indicator ofmitochondrial membrane potential.

Imaging in Human Cells and Vertebrate Models (e.g., Rat, Mouse,Zebrafish)

An optical reporter such as Arch 3 may be expressed in human embryonickidney 293 (HEK293T) cells. Fluorescence of Arch 3 in HEK 293T cells wasreadily imaged in an inverted fluorescence microscope with redillumination (λ=640 nm, 1=540 W/cm²), a high numerical apertureobjective, a Cy5 filter set, and an EMCCD camera.

FIG. 32 shows a model of Arch as a voltage sensor. pH and membranepotential can both alter the protonation of the Schiff base. The crystalstructure shown is bacteriorhodopsin; the structure of Arch has not beensolved.

FIG. 33 shows absorption (solid line) and fluorescence emission (Em,see, dashed line) spectra of purified Arch at neutral and high pH.

FIG. 34 top shows a HEK cell expressing Arch, visualized via Archfluorescence. FIG. 34 bottom shows a pixel-weight matrix regions ofvoltage-dependent fluorescence. Scale bar 10 μm.

Fluorescence of Arch 3 in HEK 293 cells was readily imaged in aninverted fluorescence microscope with red illumination (lambda=640 nm,I=540 W/cm̂2), a high numerical aperture objective, a Cy5 filter set, andan EMCCD camera. The cells exhibited fluorescence predominantlylocalized to the plasma membrane (FIG. 34). Cells not expressing Archwere not fluorescent. Cells showed 17% photobleaching over a continuous10-minute exposure, and retained normal morphology during this interval.

The fluorescence of HEK cells expressing Arch was highly sensitive tomembrane potential, as determined via whole-cell voltage clamp. Wedeveloped an algorithm to combine pixel intensities in a weighted sumsuch that the output, was a nearly optimal estimate of membranepotential V determined by conventional electrophysiology. FIG. 34 showsan example of a pixel-weight matrix, indicating that thevoltage-sensitive protein was localized to the cell membrane;intracellular Arch contributed fluorescence but no voltage-dependentsignal.

FIG. 35 shows fluorescence of Arch as a function of membrane potential.The fluorescence was divided by its value at −150 mV. The fluorescenceincreased by a factor of 2 between −150 mV and +150 mV, with a nearlylinear response throughout this range (FIG. 35). The response offluorescence to a step in membrane potential occurred within the 500micro s time resolution of our imaging system on both the rising andfalling edge.

FIG. 36 shows dynamic response of Arch to steps in membrane potentialbetween −70 mV and +30 mV. The overshoots on the rising and fallingedges were an artifact of electronic compensation circuitry. Data werean average of 20 cycles. Inset shows that step response occurred in lessthan the 0.5 ms resolution of the imaging system. The cells exhibitedfluorescence predominantly localized to the plasma membrane (FIG. 34).Cells not expressing Arch 3 were not fluorescent. Cells showed 17%photobleaching over a continuous 10-minute exposure, and retained normalmorphology during this interval. Application of a sinusoidally varyingmembrane potential led to sinusoidally varying fluorescence; at f=1 kHz,the fluorescence oscillations retained 55% of their low-frequencyamplitude (FIG. 37). Arch reported voltage steps as small as 10 mV, withan accuracy of 625 micro V/(Hz)̂(1/2) over timescales <12 s (FIG. 38).Over longer timescales laser power fluctuations and cell motion degradedthe accuracy.

FIG. 37 shows sensitivity of Arch 3 WT to voltage steps of 10 mV.Whole-cell membrane potential determined via direct voltage recording,V, (bolded black line, showing step-like line on the graph) and weightedArch 3 fluorescence, {circumflex over (V)}_(FL), (solid narrower lineshowing serrations on the graph).

FIG. 38 shows that Arch 3 reports action potentials without exogenousretinal. We made an image of 14 day in vitro (DIV) hippocampal neuronimaged via Arch 3 fluorescence with no exogenous retinal. Electrical(bolded solid black line) and fluorescence (non-bolded line, showingserrated line in the graph) records of membrane potential from theneuron during a current pulse. Action potentials are clearly resolved.

Drug Screens

A recent article reported that “Among the 100 top-selling drugs, 15 areion-channel modulators with a total market value of more than $15billion.” See Molokanova & Savchenko, 2008, Bright future of opticalassays for ion channel drug discovery, Drug Discov Today 13:14-22.However, searches for new ion-channel modulators are limited by theabsence of good indicators of membrane potential. See Przybylo et al.,2010, Fluorescence techniques for determination of the membranepotentials in high throughput screening, J Fluoresc 20(6):1139-1157. Insome embodiments, the optical reporters described herein are used tomeasure or monitor membrane potential changes in response to a candidateion channel modulator. Such screening methods can be performed in a highthroughput manner by simultaneously screening multiple candidate ionchannel modulators in cells.

The constructs disclosed in the present application can be used inmethods for drug screening, e.g., for drugs targeting the nervoussystem. In a culture of cells expressing specific ion channels, one canscreen for agonists or antagonists without the labor of applying patchclamp to cells one at a time. In neuronal cultures one can probe theeffects of drugs on action potential initiation, propagation, andsynaptic transmission. Application in human iPSC-derived neurons willenable studies on genetically determined neurological diseases, as wellas studies on the response to environmental stresses (e.g. anoxia).

Similarly, the optical voltage sensing using the constructs providedherein provides a new and much improved method to screen for drugs thatmodulate the cardiac action potential and its intercellular propagation.These screens will be useful both for determining safety of candidatedrugs and to identify new cardiac drug leads. Identifying drugs thatinteract with the hERG channel is a particularly promising directionbecause inhibition of hERG is associated with ventricular fibrillationin patients with long QT syndrome. Application in human iPSC-derivedcardiomyocytes will enable studies on genetically determined cardiacconditions, as well as studies on the response to environmental stresses(e.g. anoxia).

Additionally, the constructs of the present invention can be used inmethods to study development and wound healing. The role of electricalsignaling in normal and abnormal development, as well as tissue repair,is poorly understood. Voltage-indicating protein reporters (aka GEVIs)enable studies of voltage dynamics over long times in developing orhealing tissues, organs, and organisms, and lead to drugs that modulatethese dynamics.

In yet another embodiment, the invention provides methods to screen fordrugs that affect membrane potential of mitochondria. Mitochondria playan essential role in ageing, cancer, and neurodegenerative diseases.Currently there is no good probe for mitochondrial membrane potential.Optical reporters provide such a probe, enabling searches for drugs thatmodulate mitochondrial activity.

Prior to optical reporters, no measurement of membrane potential hadbeen made in any intact prokaryote. The PROPS voltage indicator enabledthe discovery that bacteria have complex electrical dynamics. Opticalreporters may provide screens for drugs that modulate theelectrophysiology of a wide range of medically, industrially, andenvironmentally significant microorganisms. For instance, we found thatelectrical activity is correlated with efflux pumping in E. coli.

Changes in membrane potential are also associated with activation ofmacrophages. However, this process is poorly understood due to thedifficulty in applying patch clamp to motile cells. Voltage indicatingproteins enable studies of the electrophysiology of macrophages andother motile cells, including sperm cells for fertility studies. Thusthe voltage indicating proteins of the invention can be used in methodsto screen for drugs or agents that affect, for example, immunity andimmune diseases, as well as fertility.

The examples describe expression of voltage indicating proteins in rathippocampal neurons and human iPSC-derived neurons. In all cell types,single action potentials (APs) were readily observed.

For example, in one embodiment, the invention provides a method whereinthe cell expressing a microbial rhodopsin is further exposed to astimulus capable of or suspected to be capable of changing membranepotential.

Stimuli that can be used include candidate agents, such as drugcandidates, small organic and inorganic molecules, larger organicmolecules and libraries of molecules and any combinations thereof. Onecan also use a combination of a known drug, such as an antibiotic with acandidate agent to screen for agents that may increase the effectivenessof the one or more of the existing drugs, such as antibiotics.

The methods of the invention are also useful for vitro toxicityscreening and drug development. For example, using the methods describedherein one can make a human cardiomyocyte from induced pluripotent cellsthat stably expresses a modified Archaerhodopsin wherein the protonpumping activity is substantially reduced or abolished. Such cells areparticularly useful for in vitro toxicity screening in drug development.

Multimodal Sensing/Multiplexing

Membrane potential is only one of several mechanisms of signaling withincells. One may correlate changes in membrane potential with changes inconcentration of other species, such as Ca++, H+ (i.e. pH), Na+, ATP,cAMP. We constructed fusions of Arch with pHluorin (a fluorescent pHindicator) and GCaMP6f (a fluorescent Ca++ indicator). One can also usefusions with other protein-based fluorescent indicators to enable otherforms of multimodal imaging using the concept as taught herein.Concentration of ions such as sodium, potassium, chloride, and calciumcan be simultaneously measured when the nucleic acid encoding themicrobial rhodopsin is operably linked to or fused with an additionalfluorescent ion sensitive indicator.

Additional fluorescent proteins may be included. The term “additionalfluorescent molecule” refers to fluorescent proteins other thanmicrobial rhodopsins. Such molecules may include, e.g., greenfluorescent proteins and their homologs.

Fluorescent proteins that are not microbial rhodopsins are well knownand commonly used, and examples can be found, e.g., in a review Wachter,2006, The Family of GFP-Like Proteins: Structure, Function, Photophysicsand Biosensor Applications. Introduction and Perspective, Photochem andPhotobiol 82(2):339-344. Also, Shaner et al., 2005, A guide to choosingfluorescent proteins, Nat Meth 2:905-909 provides examples of additionaluseful fluorescent proteins.

One can combine imaging of voltage indicating proteins with otherstructural and functional imaging, of e.g. pH, calcium, or ATP. One mayalso combine imaging of voltage indicating proteins with optogeneticcontrol of membrane potential using e.g. channelrhodopsin,halorhodopsin, and Archaerhodopsin. If optical measurement and controlare combined in a feedback loop, one can perform all-optical patch clampto probe the dynamic electrical response of any membrane.

The invention provides high-throughput methods of characterizing cells.Robotics and custom software may be used for screening large librariesor large numbers of conditions which are typically encountered in highthroughput drug screening methods.

Measurement Methodologies

The spectroscopic states of microbial rhodopsins are typicallyclassified by their absorption spectrum. However, in some cases there isinsufficient protein in a single cell to detect spectral shifts viaabsorbance alone. Any of the following several optical imagingtechniques can be used to probe other state-dependent spectroscopicproperties.

a) Fluorescence

It was found that many microbial rhodopsin proteins and their mutantsproduce measurable fluorescence. For example, fluorescence of anArch-based reporter may be excited by light with a wavelength betweenwavelength of 500 and 650 nm, and emission is peaked at 710 nm. The rateof photobleaching of the reporter decreases at longer excitationwavelengths, so one preferable excitation wavelength is in the redportion of the spectrum, near 633 nm. These wavelengths are further tothe red than the excitation and emission wavelengths of any otherfluorescent protein, a highly desirable property for in vivo imaging.Preferably, the fluorescence of the reporter shows negligiblephotobleaching, in stark contrast to all other known fluorophores. Whenexcited at 633 nm, the reporter and GFP emit a comparable numbers ofphotons prior to photobleaching. Thus microbial rhodopsins constitute anew class of highly photostable, membrane-bound fluorescent markers. Itmay be found that fluorescence of the reporter is sensitive to the stateof protonation of the Schiff base in that the protonated formpreferentially fluoresces. Thus voltage-induced changes in protonationenhance changes in fluorescence. In some embodiments, the fluorescenceof the reporter is detected using e.g., a fluorescent microscope, afluorescent plate reader, FACS sorting of fluorescent cells, etc.

b) Electrochromic Fluorescence Resonance Energy Transfer (eFRET)

FRET is a useful tool to quantify molecular dynamics in biophysics andbiochemistry, such as protein-protein interactions, protein-DNAinteractions, and protein conformational changes. For monitoring thecomplex formation between two molecules (e.g., retinal and microbialrhodopsin), one of them is labeled with a donor and the other with anacceptor, and these fluorophore-labeled molecules are mixed. When theyare dissociated, the donor emission is detected upon the donorexcitation. On the other hand, when the donor and acceptor are inproximity (1-10 nm) due to the interaction of the two molecules, theacceptor emission is predominantly observed because of theintermolecular FRET from the donor to the acceptor.

A fluorescent molecule appended to a microbial rhodopsin can transferits excitation energy to the retinal, but only if the absorptionspectrum of the retinal overlaps with the emission spectrum of thefluorophore. Changes in the absorption spectrum of the retinal lead tochanges in the fluorescence brightness of the fluorophore. To performelectrochromic FRET, a fluorescent protein is fused with the microbialrhodopsin voltage sensor, and the fluorescence of the protein ismonitored. This approach has the advantage over direct fluorescence thatthe emission of fluorescent proteins is far brighter than that ofretinal, but the disadvantage of being an indirect readout, with smallerfractional changes in fluorescence.

In some embodiments, voltage-induced changes in the absorption spectrumof microbial rhodopsins are detected using electrochromic FRET.

c) Rhodopsin Optical Lock-in Imaging (ROLI)

The absorption spectrum of many of the states of retinal is temporarilychanged by a brief pulse of light. In ROLI, periodic pulses of a “pump”beam are delivered to the sample. A second “probe” beam measures theabsorbance of the sample at a wavelength at which the pump beam inducesa large change in absorbance. Thus the pump beam imprints a periodicmodulation on the transmitted intensity of the probe beam. Theseperiodic intensity changes are detected by a lock-in imaging system. Incontrast to conventional absorption imaging, ROLI providesretinal-specific contrast. Modulation of the pump at a high frequencyallows detection of very small changes in absorbance.

In some embodiments, the voltage-induced changes in the absorptionspectrum of a microbial rhodopsin are detected using rhodopsin opticallock-in imaging.

d) Raman

Raman spectroscopy is a technique that can detect vibrational,rotational, and other low-frequency modes in a system. The techniquerelies on inelastic scattering of monochromatic light (e.g., a visiblelaser, a near infrared laser or a near ultraviolet laser). Themonochromatic light interacts with molecular vibrations, phonons orother excitations in the system, resulting in an energy shift of thelaser photons. The shift in energy provides information about the phononmodes in the system.

Retinal in microbial rhodopsin molecules is known to have a strongresonant Raman signal. This signal is dependent on the electrostaticenvironment around the chromophore, and therefore is sensitive tovoltage.

In some embodiments, voltage-induced changes in the Raman spectrum ofmicrobial rhodopsins are detected using Raman microscopy.

e) Second Harmonic Generation (SHG)

Second harmonic generation, also known in the art as “frequencydoubling” is a nonlinear optical process, in which photons interactingwith a nonlinear material are effectively “combined” to form new photonswith twice the energy, and therefore twice the frequency and half thewavelength of the initial photons.

SHG signals have been observed from oriented films of bacteriorhodopsinin cell membranes. SHG is an effective probe of the electrostaticenvironment around the retinal in optical voltage sensors. Furthermore,SHG imaging involves excitation with infrared light which penetratesdeep into tissue. Thus SHG imaging can be used for three-dimensionaloptical voltage sensing using the optical reporters described herein.

In some embodiments, voltage-induced changes in the second harmonicspectrum of microbial rhodopsins are detected using SHG imaging.

f) Photothermal Imaging

Photothermal imaging senses the change in refractive index in a mediumarising from a change in temperature, where the change in temperature isinduced by optical absorption. In photothermal imaging, a “pump” beam oflight is absorbed by a sample and generates local heating. A second“probe” beam of light, at a wavelength that is not absorbed by thesample, propagates through the sample. Temperature-induced changes inthe optical path length are detected by one of several opticalconfigurations, e.g. Schlieren imaging or differential interferencecontrast (DIC) microscopy.

In some embodiments, photothermal imaging is used to detectvoltage-induced changes in the absorption spectrum of a microbialrhodopsin.

Chromophore

In the wild, microbial rhodopsins contain a bound molecule of retinalwhich serves as the optically active element. These proteins will alsobind and fold around many other chromophores with similar structure, andpossibly preferable optical properties. Analogues of retinal with lockedrings cannot undergo trans-cis isomerization, and therefore have higherfluorescence quantum yields (Brack et al., Picosecond time-resolvedadsorption and fluorescence dynamics in the artificial bacteriorhodopsinpigment BR6.11, Biophys. J. 65(2):964-972). Analogues of retinal withelectron-withdrawing substituents have a Schiff base with a lower pKathan natural retinal and therefore may be more sensitive to voltage(Sheves et al., 1986, Controlling the pKa of the bacteriorhodopsinSchiff base by use of artificial retinal analogs, PNAS 83(10):3262-3266;Rousso et al., 1995, pKa of the protonated Schiff base and asparatic 85in the Bacteriorhodopsin binding site is controlled by a specificgeometry between the two resdidues, Biochemistry 34(37):12059-12065).Covalent modifications to the retinal molecule may lead to opticalvoltage sensors with significantly improved optical properties andsensitivity to voltage.

Advantages of the Methods and Compositions Described Herein

Key figures of merit for an optical voltage sensor are its responsespeed and its sensitivity (fractional change in fluorescence per 100 mVchange in membrane potential). FIG. 4 compares these attributes forprevious protein-based fluorescent voltage indicators and thosecontemplated herein. Additional important attributes include the abilityto target the indicator to a particular cell type or sub-cellularstructure, photostability, and low phototoxicity.

Previous protein-based efforts focused on fusing one or more fluorescentproteins to transmembrane voltage sensing domains. A change in voltageinduces a conformational change in the voltage sensing domain, whichmoves the fluorescent proteins, and changes their fluorescence. Thereliance on conformational motion of multiple large protein domainsmakes these approaches unavoidably slow. Furthermore, the conformationalshifts of most voltage sensing domains are small, leading to smallchanges in fluorescence.

The most sensitive indicators from the VSFP 2.x family have a change influorescence of ΔF/F=10% per 100 mV. VSFP 2.x proteins respond inapproximately 100 milliseconds, far too slow to detect a 1 ms actionpotential in a neuron (Perron et al., 2009, Second and third generationvoltage-sensitive fluorescent proteins for monitoring membranepotential, Front Mol Neurosci 2:5; Mutoh et al., 2009,Spectrally-resolved response properties of the three most advanced FRETbased fluorescent protein voltage probes, PLoS One 4:e4555). The SPARCfamily of voltage sensors has a 1 ms response time, but shows afluorescence change of <1% per 100 mV (Baker et al., 2007, Threefluorescent protein voltage sensor exhibit low plasma membranseexpression in mammalian cells, J. Neurosci. Methods 161(1):32-38; Ataka& Pieribone, 2002, A genetically targetable fluorescent probe of channelgating with rapid kinetics, Biophys J 82(1 Pt 1):509-516). The mostsensitive voltage-sensitive fluorescent proteins are the ArcLightproteins, which show a voltage sensitivity of ΔF/F=−32% per 100 mV.ArcLight and related probes are described in Jin et al., 2012, Singleaction potentials and subthreshold electrical events imaged in neuronswith a fluorescent protein voltage probe, Neuron 75(5):779-785. However,the ArcLight proteins have a slow response, with half-response times ofapproximately 100 ms at room temperature. The ASAP1 protein offers themost promising combination of sensitivity and speed, with of ΔF/F=−29%per 100 mV and a half-response time of approximately 2 ms. Prior to thepresent study described herein, two decades of research on fluorescentvoltage sensors had not yet yielded a protein that could signalindividual neuronal action potentials in vivo.

Some organic dyes show voltage-sensitive fluorescence. These lipophilicmolecules incorporate into the cell membrane where voltage leads toshifts in conformation or electronic energy levels and thereby tochanges in optical properties. These molecules respond quickly (lessthan 1 ms, typically), and have sensitivities as large as 34% per 100mV, but cannot be targeted, are often difficult to deliver, and arehighly toxic (Krauthamer et al., 1991, Action potential-inducedfluorescence changes resolved with an optical fiber carrying excitationlight, J. Fluoresc. 1(4):207-213; Fromherz et al., 2008, ANNINE-6plus, avoltage-sensitive dye with good solubility, strong membrane binding andhigh sensitivity, Eur Biophys J 37(4):509-514; Sjulso & Miesenbock,2008, Rational optimization and imaging in vivo of a genetically encodedoptical voltage reporter, J Neurosci 28(21):5582-93), see e.g. U.S. Pat.No. 6,991,910 to Adorante, U.S. Pat. No. 6,107,066 to Tsien; U.S. Pat.No. 5,661,035 to Tsien; and U.S. Pub. 2014/0093907 to Miller). None ofthese optical voltage sensors employs a microbial rhodopsin protein thatis configured to run “backwards” to convert changes in membranepotential into changes in an optically detectable signal.

The approach to optical voltage sensing described herein is differentfrom previous efforts. As described herein a protein is used that has astrong electro-optical coupling in the wild. Microbial rhodopsins in thewild serve to transduce sunlight into a membrane potential. The opticalvoltage sensors described herein use this function in reverse,transducing a membrane potential into a readily detectable opticalsignal. As FIG. 4 shows, suitable microbial rhodopsin voltage sensorsare provided.

9. Systems of the Invention

FIG. 39 presents a system 1101 useful for performing methods of theinvention. Results from a lab (e.g., transformed, converted patientcells) are loaded into imaging instrument 501. Imaging instrument 501 isoperably coupled to an analysis system 1119, which may be a PC computeror other device that includes a processor 125 coupled to a memory 127. Auser may access system 1101 via PC 1135, which also includes a processor125 coupled to a memory 127. Analytical methods described herein may beperformed by any one or more processor 125 such as may be in analysissystem 1119, PC 1135, or server 1139, which may be provided as part ofsystem 1101. Server 1139 includes a processor 125 coupled to a memory127 and may also include optional storage system 1143. Any of thecomputing device of system 1101 may be communicably coupled to oneanother via network 1131. Any, each, or all of analysis system 1119, PC1135, and server 1139 will generally be a computer. A computer willgenerally include a processor 125 coupled to a memory 127 and at leastone input/output device.

A processor 125 will generally be a silicon chip microprocessor such asone of the ones sold by Intel or AMD. Memory 127 may refer to anytangible, non-transitory memory or computer readable medium capable ofstoring data or instructions, which—when executed by a processer125—cause components of system 1101 to perform methods described herein.Typical input/output devices may include one or more of a monitor,keyboard, mouse, pointing device, network card, Wi-Fi card, cellularmodem, modem, disk drive, USB port, others, and combinations thereof.Generally, network 1131 will include hardware such as switches, routers,hubs, cell towers, satellites, landlines, and other hardware such asmakes up the Internet.

INCORPORATION BY REFERENCE

References and citations to other documents, such as patents, patentapplications, patent publications, journals, books, papers, webcontents, have been made throughout this disclosure. All such documentsare hereby incorporated herein by reference in their entirety for allpurposes.

EQUIVALENTS

Various modifications of the invention and many further embodimentsthereof, in addition to those shown and described herein, will becomeapparent to those skilled in the art from the full contents of thisdocument, including references to the scientific and patent literaturecited herein. The subject matter herein contains important information,exemplification and guidance that can be adapted to the practice of thisinvention in its various embodiments and equivalents thereof.

What is claimed is:
 1. A method for evaluating the function of a neuron,the method comprising: providing a first neuron that includes an opticalreporter of electrical activity and a second neuron that is incommunication with the first neuron via at least one synapse; obtaininga signal from the optical reporter in response to a stimulation of thesecond neuron; and evaluating the signal in order to determine afunctional property of the neuron.
 2. The method of claim 1, wherein theoptical reporter of electrical activity comprises a microbial rhodopsinwith one or more mutations.
 3. The method of claim 1, wherein the secondneuron includes an actuator of electrical activity.
 4. The method ofclaim 3, wherein the actuator of electrical activity is a protein. 5.The method of claim 4, wherein the protein is a channelrhodopsin.
 6. Themethod of claim 4, wherein characterizing the neuron comprisesevaluating a response to exposure to a compound.
 7. The method of claim4, wherein evaluating the signal comprises using a computer system tocharacterize an AP waveform of the first neuron.
 8. The method of claim4, wherein the optical reporter of electrical activity comprises amicrobial rhodopsin with one or more mutations.
 9. The method of claim 4wherein the optical reporter of electrical activity is part of a fusionprotein that also includes a fluorescent Ca++ indicator.
 10. The methodof claim 9, wherein the fluorescent Ca++ indicator is GCaMP6f.
 11. Amethod for evaluating the function of a cardiomyocyte, the methodcomprising: providing a first cardiomyocyte that includes an opticalreporter of electrical activity and a second cardiomyocyte that is incommunication with the first cardiomyocyte via at least one gapjunction; obtaining a signal from the optical reporter in response to astimulation of the second cardiomyocyte; and evaluating the signal inorder to determine a functional property of the cardiomyocyte.
 12. Themethod of claim 11, wherein the optical reporter of electrical activitycomprises a microbial rhodopsin with one or more mutations.
 13. Themethod of claim 11, wherein the second cardiomyocyte includes anactuator of electrical activity.
 14. The method of claim 13, wherein theactuator of electrical activity is a protein.
 15. The method of claim14, wherein the protein is a channelrhodopsin.
 16. The method of claim14, wherein characterizing the cardiomyocyte comprises evaluating aresponse to exposure to a compound.
 17. The method of claim 14, whereinevaluating the signal comprises using a computer system to characterizean AP waveform of the first cardiomyocyte.
 18. The method of claim 14,wherein the optical reporter of electrical activity comprises amicrobial rhodopsin with one or more mutations.
 19. The method of claim14 wherein the optical reporter of electrical activity is part of afusion protein that also includes a fluorescent Ca++ indicator.
 20. Themethod of claim 19, wherein the fluorescent Ca++ indicator is GCaMP6f.